Fusion proteins and their use in the diagnosis and treatment of leishmaniasis

ABSTRACT

The present invention relates generally to a fusion protein made from a synthetic gene construct comprising of elements derived from the  Leishmania  antigens K26, K39, and K9. The fusion protein is particularly useful in the diagnosis of leishmaniasis, particularly visceral leishmaniasis in animals such as humans and dogs.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit under 35 U.S.C. §119(e) of U.S.Provisional Patent Application No. 61/078,255 filed Jul. 3, 2008, wherethis provisional application is incorporated herein by reference in itsentirety.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 480239_(—)406_SEQUENCE_LISTING.txt. The textfile is 53 KB, was created on Jul. 2, 2009, and is being submittedelectronically via EFS-Web, concurrent with the filing of thespecification.

BACKGROUND

1. Technical Field

The present invention relates generally to fusion proteins made from asynthetic chimeric gene construct comprising sequences from K26, K39,and K9 and their use in the diagnosis and treatment of leishmaniasis.

2. Description of the Related Art

Leishmania organisms are intracellular protozoan parasites ofmacrophages that cause a wide range of clinical diseases in humans anddomestic animals, primarily dogs. In some infections, the parasite maylie dormant for many years. In other cases, the host may develop one ofa variety of forms of leishmaniasis. For example, the disease may beasymptomatic or may be manifested as sub-clinical visceralleishmaniasis, which is characterized by mild symptoms of malaise,diarrhea and intermittent hepatomegaly. Patients with sub-clinical orasymptomatic disease usually have low antibody titers, making thedisease difficult to detect with standard techniques. Alternatively,leishmaniasis may be manifested as a cutaneous disease, which is asevere medical problem but is generally self-limiting, or as a highlydestructive mucosal disease, which is not self-limiting. Finally, andmost seriously, the disease may be manifested as an acute visceralinfection involving the spleen, liver and lymph nodes, which, if leftuntreated, is generally a fatal disease. Symptoms of acute visceralleishmaniasis include hepatosplenomegaly, fever, leukopenia, anemia andhypergammaglobulinemia.

Three main clinical variants of this disease are known: cutaneous,mucocutaneous, and visceral. Cutaneous leishmaniasis can manifest itselfas a single skin ulceration at the site of the sandfly bite appearingsoon after infection or months later as disseminated lesions.Mucocutaneous syndrome develops as the cutaneous form, but progressesmonths or years later to lesions of the mouth, nose, or pharynx. Themajor long-term effects of cutaneous and mucocutaneous disease arescarring. Visceral leishmaniasis has an incubation period of 3-6 monthsand involves the reticuloendothelial system.

Clinical manifestations of visceral leishmaniasis include enlargement ofthe liver and spleen, fever, anemia, and weight loss. In the absence oftreatment, symptomatic visceral disease often ends in death. In recentyears, the coexistence of HIV and Leishmania species causing visceraldisease has resulted in several hundreds of cases of dually infectedindividuals (Berman, J. D., (1997) Clin. Infect. Dis. 24:684). The WorldHealth Organization recently estimated in 2000 that leishmaniasisaffects people in 88 countries, with 350 million at risk of contractingthe disease and about two million new cases each year. The devastatingimpact of this disease is exemplified by the recent epidemic of visceralleishmaniasis in the Sudan, which claimed an estimated 100,000 lives(Seaman, J., et al. (1996) Int. J. Epidemiol. 25:862). This disease isfrequently a threat in military operations, as demonstrated by theoutbreak of viscerotropic leishmaniasis during the Gulf War (Magill, J.,et al. (1993) N Engl J Med 328:1383).

Leishmaniasis is a serious problem in much of the world, includingBrazil, China, East Africa, India and areas of the Middle East. Thedisease is also endemic in the Mediterranean region, including southernFrance, Italy, Greece, Spain, Portugal and North Africa. The number ofcases of leishmaniasis has increased dramatically in the last 20 years,and millions of cases of this disease now exist worldwide. About 2million new cases are diagnosed each year, 25% of which are visceralleishmaniasis. There are, however, no vaccines or effective treatmentscurrently available.

Leishmaniasis is caused by several species of Leishmania. Theseunicellular organisms of the order Kinetoplastida are related totrypanosomes, the causative organisms of Sleeping Sickness in Africa andChagas' disease in South America. Leishmania parasites commonly exist intwo distinct forms, the motile promastigote of the insect vector and thesessile amastigote present in the mammalian host. Promastigotes aretransmitted to humans by the bite of infected phlebotomine sandflies,which are found throughout the world's inter-tropical and temperateregions. Upon delivery into the mammalian host, promastigotes infectmacrophages of the reticuloendothelial system and transform intoamastigotes.

Accurate diagnosis of leishmaniasis is frequently difficult to achieve.There are 20 species of Leishmania that infect humans, including L.donovani, L. chagasi, L. infantum, L. major, L. amazonensis, L.braziliensis, L. panamensis, L. mexicana, L. tropica, and L. guyanensis,and there are no distinctive signs or symptoms that unambiguouslyindicate the presence of Leishmania infection. Parasite detectionmethods have been used, but such methods are neither sensitive norclinically practical. Current skin tests typically use whole or lysedparasites. Such tests are generally insensitive, irreproducible andprone to cross-reaction with a variety of other diseases. In addition,the preparations employed in such tests are often unstable.

Thus, there is a need for improved methods for the detection ofLeishmania infection. For example, there is a need in the art for moresensitive and specific methods for detecting Leishmania infection, andfor identifying those asymptomatic Leishmania infections that are likelyto progress to acute visceral infections. The present invention fulfillsthese needs and further provides other related advantages.

BRIEF SUMMARY

The present invention relates generally to compositions comprising atleast two heterologous Leishmania antigens, fusion polypeptidescomprising the antigens, and polynucleotides encoding the antigens andfusions, wherein the Leishmania antigens are selected from K39, K26,and/or K9. The present invention also relates methods of using thepolypeptides and polynucleotides of the invention in the diagnosis,treatment, and prevention of leishmaniasis. The antigens of theinvention, when employed in combination and/or as fusion polypeptides orpolynucleotides as described herein, offer improved and unexpectedadvantages, and are particularly useful in the context of leishmaniasisdiagnostics and vaccine development.

Therefore, according to one embodiment, the present invention providesan isolated fusion polypeptide comprising two or more Leishmaniaantigens selected from the group consisting of K26, K39, and K9, orimmunogenic portions or variants thereof. In a related embodiment, anisolated fusion polypeptide comprises at least all three of the aboveLeishmania antigens, wherein at least one of the antigens is isolatedfrom Leishmania donovani.

In particular embodiments, fusion polypeptides of the invention comprisea first immunogenic portion comprising at least residues 142-267 of K26(SEQ ID NO: 5); a second immunogenic portion comprising at leastresidues 2110-2343 of K39 (SEQ ID NO: 6); and a third immunogenicportion comprising at least residues 1-399 of K9 (SEQ ID NO: 7). In moreparticular embodiments, a fusion polypeptide of the present inventioncomprises an amino acid sequence as set forth in amino acid residues10-262 of SEQ ID NO: 8. In certain embodiments, the fusion polypeptidecomprises an N-terminal amino acid sequence of MHHHHHHTS (SEQ ID NO:21). In certain other embodiments, the fusion polypeptide comprises anamino acid sequence as set forth in SEQ ID NO: 8.

In various embodiments, the present invention provides isolatedpolynucleotides encoding a fusion polypeptide of the present invention.

The present invention also provides an isolated antibody, or antigenbinding fragment that specifically bind a fusion polypeptide asdescribed herein throughout.

In another embodiment, the invention contemplates, pharmaceuticalcomposition comprising a fusion polypeptide of the present invention, oran isolated antibody or antigen binding fragment recognizing a fusionpolypeptide of the present invention, or a polynucleotide encoding afusion polypeptide of the present invention, in combination with aphysiologically acceptable carrier.

In a related embodiment, the present invention contemplates vaccinecompositions comprising a fusion polypeptide of the present invention,or an isolated antibody or antigen binding fragment recognizing a fusionpolypeptide of the present invention, or a polynucleotide encoding afusion polypeptide of the present invention, in combination with anon-specific immune response enhancer.

In particular embodiments, the present invention provides methods todetect asymptomatic or sub-clinical Leishmania infection in a biologicalsample selected from the group consisting of sera, blood, and saliva,comprising: contacting a biological sample with a fusion polypeptidecomprising two or more Leishmania antigens selected from the groupconsisting of K26, K39, and K9, or immunogenic portions thereof; anddetecting in the biological sample the presence of antibodies that bindto the fusion polypeptide, thereby detecting asymptomatic sub-clinicalor active Leishmania infection in the biological sample. In moreparticular embodiments, a method to detect asymptomatic or sub-clinicalLeishmania infection in a biological sample employs a fusion polypeptidecomprising: a first immunogenic portion comprising at least residues142-267 of K26 (SEQ ID NO: 5); a second immunogenic portion comprisingat least residues 2110-2343 of K39 (SEQ ID NO: 6); and a thirdimmunogenic portion comprising at least residues 1-399 of K9 (SEQ ID NO:7). In yet more particular embodiments, the fusion polypeptide comprisesan amino acid sequence as set forth in residues 10-262 of SEQ ID NO: 8.In yet more particular related embodiments, the fusion polypeptidefurther comprises an N-terminal amino acid sequence of MHHHHHHTS (SEQ IDNO: 21).

In related embodiments, methods for detecting asymptomatic orsub-clinical Leishmania infection in a biological sample use a fusionpolypeptide bound to a solid support, wherein the solid support maycomprise, for example, nitrocellulose, latex or a plastic material. Incertain embodiments, the methods further comprise i) removing unboundsample from a solid support; ii) adding a detection reagent to the solidsupport; and iii) determining the level of detection reagent bound tothe solid support, relative to a predetermined cutoff value, therebydetecting asymptomatic or sub-clinical Leishmania infection in thebiological sample.

In various embodiments, the present invention provides methods ofidentifying a patient afflicted with asymptomatic or sub-clinicalvisceral leishmaniasis that is likely to develop acute visceralleishmaniasis, comprising: contacting a biological sample obtained froma patient afflicted with asymptomatic or sub-clinical visceralleishmaniasis with a first polypeptide that is a fusion polypeptidecomprising two or more Leishmania antigens selected from the groupconsisting of K26, K39, and K9, or immunogenic portions thereof, thebiological sample being selected from the group consisting of sera,blood, and saliva; and independently contacting the biological samplewith a second polypeptide comprising an amino acid sequence as set forthin SEQ ID NOs: 6, 10 or 11; and detecting in the sample the presence ofantibodies that bind to at least one of the first and secondpolypeptides, thereby identifying a patient afflicted with asymptomaticor sub-clinical visceral leishmaniasis that is likely to develop acutevisceral leishmaniasis.

In particular embodiments, the methods of identifying a patientafflicted with asymptomatic or sub-clinical visceral leishmaniasis thatis likely to develop acute visceral leishmaniasis employ a fusionpolypeptide comprising a first immunogenic portion comprising at leastresidues 142-267 of K26 (SEQ ID NO: 5); a second immunogenic portioncomprising at least residues 2110-2343 of K39 (SEQ ID NO: 6); and athird immunogenic portion comprising at least residues 1-399 of K9 (SEQID NO: 7). In yet more particular embodiments, the fusion polypeptidecomprises an amino acid sequence as set forth in amino acid residues10-262 of SEQ ID NO: 8. In yet more particular related embodiments, thefusion polypeptide further comprises an N-terminal amino acid sequenceof MHHHHHHTS (SEQ ID NO: 21).

In related embodiments, methods of identifying a patient afflicted withasymptomatic or sub-clinical visceral leishmaniasis that is likely todevelop acute visceral leishmaniasis employ a fusion polypeptide boundto a solid support, wherein the solid support may comprise, for example,nitrocellulose, latex or a plastic material. In certain relatedembodiments, the methods further comprise i) removing unbound samplefrom each solid support; ii) adding a detection reagent to each solidsupport; and iii) comparing the level of detection reagent bound to eachsolid support, relative to a predetermined cutoff value, therefromidentifying a patient afflicted with asymptomatic or sub-clinicalleishmaniasis that is likely to develop acute visceral leishmaniasis.

In particular embodiments, the detection reagent comprises a reportergroup conjugated to a binding agent. In related embodiments the bindingagent is selected from the group consisting of anti-immunoglobulin,Protein G, Protein A and lectins. In further related embodiments, thereporter group is selected from the group consisting of radioisotopes,fluorescent groups, luminescent groups, enzymes, colloidal gold, biotinand dye particles.

In various other embodiments, the present invention provides diagnostickits for detecting asymptomatic or sub-clinical visceral leishmaniasisin a biological sample. In particular embodiments, kits for detectingasymptomatic or sub-clinical visceral leishmaniasis in a biologicalsample, wherein the sample is selected from the group consisting ofsera, blood, and saliva, comprise a fusion polypeptide comprising two ormore Leishmania antigens selected from the group consisting of K26, K39,and K9, or immunogenic portions thereof; and a detection reagent.

In certain embodiments, a diagnostic kit for identifying a patientafflicted with asymptomatic or sub-clinical visceral leishmaniasis thatis likely to develop acute visceral leishmaniasis, contains a firstpolypeptide that is a fusion polypeptide comprising two or moreLeishmania antigens selected from the group consisting of K26, K39, andK9, or immunogenic portions thereof; and a second polypeptide comprisingan amino acid sequence as set forth in SEQ ID NOs: 10 or 11; and adetection reagent.

Kits of the present invention may further comprise, a detection reagentcomprising a reporter group conjugated to a binding agent and/or abinding agent is selected from the group consisting ofanti-immunoglobulin, Protein G, Protein A and lectins and/or a reportergroup is selected from the group consisting of radioisotopes,fluorescent groups, luminescent groups, enzymes, colloidal gold, biotinand dye particles.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 shows a schematic diagram of the K28 fusion gene construct, whichcomprises an N-terminal 9 amino acid motif comprising a 6× HIS tag(i.e., 6 histidine residues), polynucleotides 142-267 of an LdK26 genesequence (SEQ ID NO: 1), polynucleotides 2110-2343 of an LdK39 genesequence (SEQ ID NO: 2), and polynucleotides 1-399 of an LdK9 genesequence (SEQ ID NO: 3).

FIG. 2 shows a comparison of the ability of K28, K39, and K9 antigens todetect VL in the serum of human patients from Venezuela.

FIG. 3 shows a comparison of the ability of K9, K26, K28, and K39antigens to detect VL in the serum of canines from Venezuela.

BRIEF DESCRIPTION OF THE SEQUENCE IDENTIFIERS

SEQ ID NO: 1 represents polynucleotides 142-267 of a L. donovani K26gene sequence, which encode an immunogenic portion of the K26 antigen.

SEQ ID NO: 2 represents polynucleotides 2110-2343 of a L. donovani K39gene sequence, which encode an immunogenic portion of the K39 antigen.

SEQ ID NO: 3 represents polynucleotides 1-399 of a L. donovani K9 genesequence, which encodes an immunogenic portion and the full-length K9antigen.

SEQ ID NO: 4 represents the polynucleotide sequence of a synthetic gene,K28, which comprises an N-terminal 9 amino acid motif comprising a 6×HIS tag, the polynucleotide sequence of SEQ ID NO: 1, fused to thepolynucleotide sequence of SEQ ID NO:2, which is fused to thepolynucleotide sequence of SEQ ID NO3. The ORF begins at nucleotide 4.

SEQ ID NO: 5 represents the amino acid sequence of an immunogenicpolypeptide encoded by polynucleotides 142-267 of a L. donovani K26antigen.

SEQ ID NO: 6 represents the amino acid sequence of an immunogenicpolypeptide encoded by polynucleotides 2110-2343 of a L. donovani K39antigen.

SEQ ID NO: 7 represents the amino acid sequence of the immunogenicfull-length polypeptide encoded by polynucleotides 1-399 of a L.donovani K9 antigen.

SEQ ID NO: 8 represents the amino acid sequence of the K28 polypeptideencoded by SEQ ID NO: 4.

SEQ ID NO: 9 represents the amino acid sequence of a single repeat unitof a L. donovani K26 antigen.

SEQ ID NO: 10 represents the amino acid sequence of a single repeat unitof a L. donovani K39 antigen.

SEQ ID NO: 11 represents the amino acid sequence of a single repeat unitof a L. donovani K39 antigen.

SEQ ID NO: 12 represents a polynucleotide sequence encoding a singlerepeat unit of a L. donovani K26 antigen according to SEQ ID NO: 9.

SEQ ID NO: 13 represents a polynucleotide sequence encoding a singlerepeat unit of a L. donovani K26 antigen according to SEQ ID NO: 9.

SEQ ID NO: 14 represents a polynucleotide sequence encoding the singlerepeat unit of a L. donovani K39 antigen according to SEQ ID NO: 10.

SEQ ID NO: 15 represents a polynucleotide sequence encoding the singlerepeat unit of a L. donovani K39 antigen according to SEQ ID NO: 11.

SEQ ID NO: 16 represents the full-length polynucleotide sequence of a L.donovani K26 gene.

SEQ ID NO: 17 represents the full-length polynucleotide sequence of a L.donovani K39 gene.

SEQ ID NO: 18 represents the full-length polynucleotide sequence of a L.donovani K9 gene.

SEQ ID NO: 19 represents the amino acid sequence encoded by SEQ ID NO:16.

SEQ ID NO: 20 represents the amino acid sequence encoded by SEQ ID NO:17.

SEQ ID NO: 21 represents an illustrative N-terminal histidine tag usedin the recombinant production of polypeptides of the present invention.

DETAILED DESCRIPTION

Leishmania Antigens and Fusions Thereof

The present invention relates generally to compositions and methods ofusing Leishmania antigens. The compositions of the present inventiongenerally comprise at least two heterologous antigens or immunogenicportion thereof of a Leishmania species. Leishmania antigen sequencescan be obtained, for example, from the National Center for BiotechnologyInformation (NCBI) database for a variety of Leishmania species,including L. donovani, L. chagasi, L. infantum, L. major, L.amazonensis, L. venezuelensis, L. braziliensis, L. panamensis, L.mexicana, L. tropica, and L. guyanensis.

In one aspect, the present invention provides isolated Leishmaniapolypeptides, as described herein, including fusion polypeptides, andcompositions containing the same. Generally, a polypeptide of thepresent invention will be an isolated polypeptide and may comprise apolypeptide fragment (e.g., an antigenic/immunogenic portion), multiplepolypeptide fragments (e.g., a fusion polypeptide), or a full-lengthpolypeptide of an amino acid sequence from two or more of the Leishmaniagenes, including, but not limited to K26, K39, and/or K9. One ofordinary skill in the art would appreciate that antigenic polypeptidefragments could also be obtained from those already available in theart. Polypeptides of the invention, antigenic/immunogenic fragmentsthereof, and other variants may be prepared using conventionalrecombinant and/or synthetic techniques.

In certain embodiments, the polypeptides of the present invention arefusion polypeptides. In particular embodiments, the fusion polypeptidescomprise K26, K39, and/or K9 antigens or immunogenic portions thereof,which are antigenic/immunogenic, i.e., they react detectably within animmunoassay (such as an ELISA or T cell stimulation assay) with antiseraand/or T cells from an infected subject. Screening for immunogenicactivity can be performed using techniques well known to the skilledartisan. For example, such screens can be performed using methods suchas those described in Harlow and Lane, Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory Press, 1988. In one illustrative example,a polypeptide of the present invention (e.g., a fusion polypeptide) maybe immobilized on a solid support and contacted with patient sera toallow binding of antibodies within the sera to the immobilizedpolypeptide. Unbound sera may then be removed and bound antibodiesdetected using, for example, ¹²⁵I-labeled Protein A.

As would be recognized by the skilled artisan, the fusion polypeptidesof the present invention may comprise antigenic or immunogenic portionsor fragments of the Leishmania K26, K39, and/or K9 polypeptidesdisclosed herein. An “immunogenic portion,” as used herein, is afragment of an immunogenic polypeptide of the invention that itself isimmunologically reactive (i.e., specifically binds) with the B-cellsand/or T cell surface antigen receptors that recognize the polypeptide.Immunogenic portions may generally be identified using well knowntechniques, such as those summarized in Paul, Fundamental Immunology,5th ed., Lippincott Williams & Wilkins, 2003 and references citedtherein. Such techniques include screening polypeptides for the abilityto react with antigen-specific antibodies, antisera and/or T cell linesor clones. As used herein, antisera and antibodies are“antigen-specific” if they specifically bind to an antigen (i.e., theyreact with the protein in an immunoassay, and do not react detectablywith unrelated proteins). Such antisera and antibodies may be preparedas described herein and using well-known techniques.

In a particular embodiment, an antigenic/immunogenic portion orpolypeptide fragment of a fusion polypeptide of the present invention isa portion that reacts with antisera and/or T cells at a level that isnot substantially less than the reactivity of the full-length fusionpolypeptide (e.g., in an ELISA and/or T cell reactivity assay).Preferably, the level of immunogenic activity of theantigenic/immunogenic portion within a fusion polypeptide is at leastabout 50%, preferably at least about 70% and most preferably greaterthan about 90% of the immunogenicity for the full-length fusionpolypeptide. In some instances, preferred immunogenic portions will beidentified that have a level of immunogenic activity greater than thatof the corresponding full-length fusion polypeptide, e.g., havinggreater than about 100% or 150% or more immunogenic activity. Inparticular embodiments, the immunogenicity of the full-length fusionpolypeptide will have additive immunogenicity contributed by of each ofthe antigenic/immunogenic portions contained therein.

A fusion polypeptide of the invention may also comprise one or morepolypeptides that are immunologically reactive with T cells and/orantibodies generated against a polypeptide of the invention,particularly a polypeptide having an amino acid sequence disclosedherein, or to an immunogenic fragment or variant thereof. In particularembodiments, the polypeptide is a fusion polypeptide as describedherein.

In another embodiment of the invention, fusion polypeptides are providedthat comprise two or more polypeptides that are capable of eliciting Tcells and/or antibodies that are immunologically reactive with two ormore polypeptides described herein, or two or more polypeptides encodedby contiguous polynucleotide sequences contained in the polynucleotidesequences disclosed herein, or immunogenic fragments or variantsthereof, or to two or more polynucleotide sequences which hybridize totwo or more of these sequences under conditions of moderate to highstringency.

The present invention also provides fusion polypeptides comprisingfragments, of K26, K29, and/or K9 polypeptides, includingantigenic/immunogenic fragments comprising at least about 5, 10, 15, 20,25, 50, 75, 100, 150, 200, 250, 300, or 350 contiguous amino acids, ormore, including all intermediate lengths, of a Leishmania K26, K39,and/or K9 antigen, such as those set forth herein, or those encoded by apolynucleotide sequence set forth herein.

In another aspect, fusion polypeptides of the present invention containmultiple copies of polypeptide fragments, repeats of polypeptidefragments, or multimeric polypeptide fragments, includingantigenic/immunogenic fragments such as Leishmania K26, K39, and/or K9polypeptides, comprising at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 ormore contiguous fragments, in any order, and including all lengths of apolypeptide composition set forth herein, or those encoded by apolynucleotide sequence set forth herein. In another aspect, fusionpolypeptides of the present invention may comprise two or moreLeishmania antigen fragments as recited in SEQ ID NOs: 5-7, and 9-11. Ina related aspect, the fusion polypeptide comprises the amino acidsequence set forth in SEQ ID NO: 8 or amino acids 10-262 of SEQ ID NO:8.

In yet another aspect, the present invention provides fusionpolypeptides comprising two or more variants of the Leishmania K26, K29,and/or K9 polypeptides described herein. Polypeptide variants generallyencompassed by the present invention will typically exhibit at leastabout 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,or 99% or more identity (determined as described below), along itslength, to a polypeptide sequence set forth herein. Preferably, suchvariants will have the same, similar or improved immunological activityrelative to a native K26, K29, and/or K9 sequence.

A polypeptide “variant,” as the term is used herein, is a polypeptidethat typically differs from a polypeptide specifically disclosed herein(e.g., Leishmania K26, K29, and/or K9 polypeptides) in one or moresubstitutions, deletions, additions and/or insertions. Such variants maybe naturally occurring or may be synthetically generated, for example,by modifying one or more of the above polypeptide sequences of theinvention and evaluating their immunogenic activity as described hereinusing any of a number of techniques well known in the art.

In many instances, a variant will contain conservative substitutions. A“conservative substitution” is one in which an amino acid is substitutedfor another amino acid that has similar properties, such that oneskilled in the art of peptide chemistry would expect the secondarystructure and hydropathic nature of the polypeptide to be substantiallyunchanged. As described above, modifications may be made in thestructure of the polynucleotides and polypeptides of the presentinvention and still obtain a functional molecule that encodes a variantor derivative polypeptide with desirable characteristics, e.g., withimmunogenic characteristics. When it is desired to alter the amino acidsequence of a polypeptide to create an equivalent, or even an improved,immunogenic variant or portion of a polypeptide of the invention, oneskilled in the art will typically change one or more of the codons ofthe encoding DNA sequence according to Table 1.

For example, certain amino acids may be substituted for other aminoacids in a protein structure without appreciable loss of interactivebinding capacity with structures such as, for example, antigen-bindingregions of antibodies or binding sites on substrate molecules. Since itis the interactive capacity and nature of a protein that defines thatprotein's biological functional activity, certain amino acid sequencesubstitutions can be made in a protein sequence, and, of course, itsunderlying DNA coding sequence, and nevertheless obtain a protein withlike properties. It is thus contemplated that various changes may bemade in the peptide sequences of the disclosed compositions, orcorresponding DNA sequences which encode said peptides withoutappreciable loss of their biological utility or activity.

TABLE 1 Amino Acids Codons Alanine Ala A GCA GCC GCG GCU Cysteine Cys CUGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA GAGPhenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine HisH CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine LeuL UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAUProline Pro P CCA CCC CCG CCU Glutamine Gln Q CAA CAG Arginine Arg R AGAAGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr TACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGGTyrosine Tyr Y UAC UAU

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, 1982, incorporated herein byreference). It is accepted that the relative hydropathic character ofthe amino acid contributes to the secondary structure of the resultantprotein, which in turn defines the interaction of the protein with othermolecules, for example, enzymes, substrates, receptors, DNA, antibodies,antigens, and the like. Each amino acid has been assigned a hydropathicindex on the basis of its hydrophobicity and charge characteristics(Kyte and Doolittle, 1982). These values are: isoleucine (+4.5); valine(+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5);methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7);serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6);histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5);asparagine (−3.5); lysine (−3.9); and arginine (−4.5).

It is known in the art that certain amino acids may be substituted byother amino acids having a similar hydropathic index or score and stillresult in a protein with similar biological activity, i.e. still obtaina biological functionally equivalent protein. In making such changes,the substitution of amino acids whose hydropathic indices are within ±2is preferred, those within ±1 are particularly preferred, and thosewithin ±0.5 are even more particularly preferred. It is also understoodin the art that the substitution of like amino acids can be madeeffectively on the basis of hydrophilicity.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). It isunderstood that an amino acid can be substituted for another having asimilar hydrophilicity value and still obtain a biologically equivalent,and in particular, an immunologically equivalent protein. In suchchanges, the substitution of amino acids whose hydrophilicity values arewithin ±2 is preferred, those within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

As outlined above, amino acid substitutions are generally thereforebased on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, hydrophilicity, charge,size, and the like. Exemplary substitutions that take various of theforegoing characteristics into consideration are well known to those ofskill in the art and include: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine.

In addition, any polynucleotide may be further modified to increasestability in vivo. Possible modifications include, but are not limitedto, the addition of flanking sequences at the 5′ and/or 3′ ends; the useof phosphorothioate or 2′ O-methyl rather than phosphodiesteraselinkages in the backbone; and/or the inclusion of nontraditional basessuch as inosine, queosine and wybutosine, as well as acetyl- methyl-,thio- and other modified forms of adenine, cytidine, guanine, thymineand uridine.

Amino acid substitutions may further be made on the basis of similarityin polarity, charge, solubility, hydrophobicity, hydrophilicity and/orthe amphipathic nature of the residues. For example, negatively chargedamino acids include aspartic acid and glutamic acid; positively chargedamino acids include lysine and arginine; and amino acids with unchargedpolar head groups having similar hydrophilicity values include leucine,isoleucine and valine; glycine and alanine; asparagine and glutamine;and serine, threonine, phenylalanine and tyrosine. Other groups of aminoacids that may represent conservative changes include: (1) ala, pro,gly, glu, asp, gin, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile,leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. Avariant may also, or alternatively, contain nonconservative changes. Ina preferred embodiment, variant polypeptides differ from a nativesequence by substitution, deletion or addition of five amino acids orfewer. Variants may also (or alternatively) be modified by, for example,the deletion or addition of amino acids that have minimal influence onthe immunogenicity, secondary structure and hydropathic nature of thepolypeptide.

As noted above, polypeptides may comprise a signal (or leader) sequenceat the N-terminal end of the protein, which co-translationally orpost-translationally directs transfer of the protein. The polypeptidemay also be conjugated to a linker or other sequence for ease ofsynthesis, purification or identification of the polypeptide (e.g.,poly-Histidine tag (6× His), GST, MBP, TAP/TAG, FLAG epitope, MYCepitope, V5 epitope, VSV-G epitope, etc.), or to enhance binding of thepolypeptide to a solid support. For example, a polypeptide may beconjugated to an immunoglobulin Fc region.

When comparing polypeptide sequences, two sequences are said to be“identical” if the sequence of amino acids in the two sequences is thesame when aligned for maximum correspondence, as described below.Comparisons between two sequences are typically performed by comparingthe sequences over a comparison window to identify and compare localregions of sequence similarity. A “comparison window” as used herein,refers to a segment of at least about 20 contiguous positions, usually30 to about 75, 40 to about 50, in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned.

Optimal alignment of sequences for comparison may be conducted using theMegalign program in the Lasergene suite of bioinformatics software(DNASTAR, Inc., Madison, Wis.), using default parameters. This programembodies several alignment schemes described in the followingreferences: Dayhoff, M. O. (1978) A model of evolutionary change inproteins—Matrices for detecting distant relationships. In Dayhoff, M. O.(ed.) Atlas of Protein Sequence and Structure, National BiomedicalResearch Foundation, Washington D.C. Vol. 5, Suppl. 3, pp. 345-358; HeinJ. (1990) Unified Approach to Alignment and Phylogenes pp. 626-645Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.;Higgins, D. G. and Sharp, P. M. (1989) CABIOS 5:151-153; Myers, E. W.and Muller W. (1988) CABIOS 4:11-17; Robinson, E. D. (1971) Comb. Theor11:105; Santou, N. Nes, M. (1987) Mol. Biol. Evol. 4:406-425; Sneath, P.H. A. and Sokal, R. R. (1973) Numerical Taxonomy—the Principles andPractice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.;Wilbur, W. J. and Lipman, D. J. (1983) Proc. Nat'l Acad., Sci. USA80:726-730.

Alternatively, optimal alignment of sequences for comparison may beconducted by the local identity algorithm of Smith and Waterman (1981)Add. APL. Math 2:482, by the identity alignment algorithm of Needlemanand Wunsch (1970) J. Mol. Biol. 48:443, by the search for similaritymethods of Pearson and Lipman (1988) Proc. Nat'l Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT,BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package,Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.), or byinspection.

One preferred example of algorithms that are suitable for determiningpercent sequence identity and sequence similarity are the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al. (1977)Nucl. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol.215:403-410, respectively. BLAST and BLAST 2.0 can be used, for examplewith the parameters described herein, to determine percent sequenceidentity for the polynucleotides and polypeptides of the invention.Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information. For amino acid sequences,a scoring matrix can be used to calculate the cumulative score.Extension of the word hits in each direction are halted when: thecumulative alignment score falls off by the quantity X from its maximumachieved value; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, Tand X determine the sensitivity and speed of the alignment.

In one preferred approach, the “percentage of sequence identity” isdetermined by comparing two optimally aligned sequences over a window ofcomparison of at least 20 positions, wherein the portion of thepolypeptide sequence in the comparison window may comprise additions ordeletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent,or 10 to 12 percent, as compared to the reference sequences (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. The percentage is calculated by determining the number ofpositions at which the identical amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the referencesequence (i.e., the window size) and multiplying the results by 100 toyield the percentage of sequence identity.

In certain preferred embodiments of the invention, there are providedLeishmania fusion polypeptides, and polynucleotides encoding fusionpolypeptides, wherein the fusion polypeptides comprise two or more ofK26, K39, and/or K9 polypeptide sequences or immunogenic portionsthereof. Fusion polypeptide and fusion proteins refer to a polypeptidehaving at least two heterologous Leishmania sp. polypeptides, such asLeishmania donovani polypeptides, covalently linked, either directly orvia an amino acid linker. The K26, K39, and/or K9 antigenic sequencesmay, but need not, be derived from the same Leishmania species. Thepolypeptides forming the fusion protein are typically linked C-terminusto N-terminus, although they can also be linked C-terminus toC-terminus, N-terminus to N-terminus, or N-terminus to C-terminus. Thepolypeptides of the fusion protein can be in any order and may containmultiple copies of, repeats of, or multimers of each or any of thepolypeptides comprising the fusion protein. Fusion polypeptides orfusion proteins can also include conservatively modified variants,polymorphic variants, alleles, mutants, subsequences, interspecieshomologs, and immunogenic fragments of the antigens that make up thefusion protein. In particular embodiments, fusion polypeptides of thepresent invention may comprise one or more Leishmania antigen fragmentsas recited in SEQ ID NOs: 5-7, and 9-11. In a related embodiment, thefusion polypeptide comprises the amino acid sequence set forth in SEQ IDNO: 8 or amino acids 10-262 of SEQ ID NO: 8.

Antigens from other Leishmania species that correspond to Leishmaniadonovani antigens may also be used and can be identified, e.g., usingsequence comparison algorithms, as described herein, or other methodsknown to those of skill in the art, e.g., hybridization assays andantibody binding assays.

The fusion polypeptides of the invention generally comprise at least twoantigenic/immunogenic portions or fragments from K26, K39, and/or K9polypeptides as described herein, and may further comprise otherunrelated sequences, such as a sequence that assists in providing Thelper epitopes (an immunological fusion partner), preferably T helperepitopes recognized by humans, or that assists in expressing the protein(an expression enhancer) at higher yields than the native recombinantprotein. Certain preferred fusion partners are both immunological andexpression enhancing fusion partners. Other fusion partners may beselected so as to increase the solubility of the protein or to enablethe protein to be targeted to desired intracellular compartments. Stillfurther fusion partners may include affinity tags such as V5, 6× HIS,MYC, FLAG, and GST, which facilitate purification of the protein. Itwould be understood by one having ordinary skill in the art that thoseunrelated sequences may, but need not, be present in a fusionpolypeptide used in accordance with the present invention.

Fusion proteins may generally be prepared using standard techniques.Preferably, a fusion protein is expressed as a recombinant protein. Forexample, DNA sequences encoding the polypeptide components of a desiredfusion may be assembled separately, and ligated into an appropriateexpression vector. The 3′ end of the DNA sequence encoding onepolypeptide component is ligated, with or without a peptide linker, tothe 5′ end of a DNA sequence encoding the second polypeptide componentso that the reading frames of the sequences are in phase. This permitstranslation into a single fusion protein that retains the biologicalactivity of both component polypeptides.

A peptide linker sequence may be employed to separate the first andsecond polypeptide components by a distance sufficient to ensure thateach polypeptide folds into its secondary and tertiary structures, ifdesired. Such a peptide linker sequence is incorporated into the fusionprotein using standard techniques well known in the art. Certain peptidelinker sequences may be chosen based on the following factors: (1) theirability to adopt a flexible extended conformation; (2) their inabilityto adopt a secondary structure that could interact with functionalepitopes on the first and second polypeptides; and (3) the lack ofhydrophobic or charged residues that might react with the polypeptidefunctional epitopes. Preferred peptide linker sequences contain Gly, Asnand Ser residues. Other near neutral amino acids, such as Thr and Alamay also be used in the linker sequence. Amino acid sequences which maybe usefully employed as linkers include those disclosed in Maratea etal., Gene 40:39 46 (1985); Murphy et al., Proc. Natl. Acad. Sci. USA83:8258 8262 (1986); U.S. Pat. No. 4,935,233 and U.S. Pat. No.4,751,180. The linker sequence may generally be from 1 to about 50 aminoacids in length. Linker sequences are not required when the first andsecond polypeptides have non-essential N-terminal amino acid regionsthat can be used to separate the functional domains and prevent stericinterference.

The ligated DNA sequences are operably linked to suitabletranscriptional or translational regulatory elements. The regulatoryelements responsible for expression of DNA are located only 5′ to theDNA sequence encoding the first polypeptides. Similarly, stop codonsrequired to end translation and transcription termination signals areonly present 3′ to the DNA sequence encoding the second polypeptide.

Within preferred embodiments, an immunological fusion partner for use ina fusion polypeptide of the invention is derived from protein D, asurface protein of the gram-negative bacterium Haemophilus influenza B(WO 91/18926). Preferably, a protein D derivative comprisesapproximately the first third of the protein (e.g., the first N-terminal100 110 amino acids), and a protein D derivative may be lipidated.Within certain preferred embodiments, the first 109 residues of alipoprotein D fusion partner is included on the N-terminus to providethe fusion polypeptide with additional exogenous T cell epitopes and toincrease the expression level in E. coli (thus functioning as anexpression enhancer). The lipid tail ensures optimal presentation of theantigen to antigen presenting cells. Other fusion partners include thenon-structural protein from influenzae virus, NS1 (hemaglutinin).Typically, the N-terminal 81 amino acids are used, although differentfragments that include T-helper epitopes may be used. In anotherembodiment, an immunological fusion partner comprises an amino acidsequence derived from the protein known as LYTA, or a portion thereof(preferably a C-terminal portion). LYTA is derived from Streptococcuspneumoniae, which synthesizes an N-acetyl-L-alanine amidase known asamidase LYTA (encoded by the LytA gene; Gene 43:265-292 (1986)). LYTA isan autolysin that specifically degrades certain bonds in thepeptidoglycan backbone. The C-terminal domain of the LYTA protein isresponsible for the affinity to the choline or to some choline analoguessuch as DEAE. This property has been exploited for the development of E.coli C-LYTA expressing plasmids useful for expression of fusionproteins. Purification of hybrid proteins containing the C-LYTA fragmentat the amino terminus has been described (see Biotechnology 10:795-798(1992)). Within a preferred embodiment, a repeat portion of LYTA may beincorporated into fusion polypeptides of the present invention, asdescribed herein. A repeat portion is found in the C-terminal regionstarting at residue 178. A particularly preferred repeat portionincorporates residues 188-305.

In general, polypeptides and fusion polypeptides (as well as theirencoding polynucleotides) are isolated. An “isolated” polypeptide orpolynucleotide is one that is removed from its original environment. Forexample, a naturally-occurring protein is isolated if it is separatedfrom some or all of the coexisting materials in the natural system.Preferably, such polypeptides are at least about 90% pure, morepreferably at least about 95% pure and most preferably at least about99% pure. A polynucleotide is considered to be isolated if, for example,it is cloned into a vector that is not a part of the naturalenvironment.

In certain embodiments, the fusion polypeptides of the invention willcomprise at least one epitope from each of K26, K39 and K9. Epitopes maygenerally be determined by generating polypeptides containing portionsor fragments of the sequence and evaluating the reactivity of thepolypeptides with sera from Leishmania-infected individuals using, forexample, an enzyme linked immunosorbent assay (ELISA). Suitable assaysfor evaluating reactivity of a polypeptide with Leishmania-infected seraare described in more detail below. Within such representative assays,portions of the sequence that generate a signal that differentiatesbetween positive and negative sera in a manner substantially similar tothat of the full length are considered to contain an epitope. In otherwords, a portion of the antigen that contains an epitope will generate asignal indicating Leishmania infection in substantially all (i.e., atleast about 80%, and preferably at least about 90%) of the biologicalsamples for which such infection would be indicated using the fulllength antigen and will generate a signal indicating the absence ofLeishmania infection in substantially all of those samples that would benegative with the full length polypeptide.

In a related aspect, fusion polypeptides comprising epitopes of multipleLeishmania polypeptides are disclosed. In certain particularembodiments, epitopes of different Leishmania polypeptides, repeats, orvariants thereof, are joined though a peptide linkage into a singleamino acid chain. The epitopes may be joined directly (i.e., with nointervening amino acids) or may be joined by way of a linker sequence(e.g., Gly-Cys-Gly) that does not significantly alter the antigenicproperties of the epitopes. In particular embodiments the fusionpolypeptide is derived from two or more antigenic/immunogenic portionsor fragments. In another aspect, fusion polypeptides of the presentinvention may comprise two or more Leishmania antigen fragments asrecited in SEQ ID NOs: 5-7 and 9-11. In a related aspect, the fusionpolypeptide comprises the amino acid sequence set forth in SEQ ID NO: 8or amino acid residues 10-262 of SEQ ID NO: 8.

The fusion polypeptides of this invention may be generated usingtechniques well known to those of ordinary skill in the art.Polypeptides of the present invention having fewer than about 100 aminoacids, and generally fewer than about 50 amino acids, can be synthesizedusing, for example, the Merrifield solid-phase synthesis method, whereamino acids are sequentially added to a growing amino acid chain(Merrifield, J. Am. Chem. Soc. 85:2149-2146, 1963). Equipment forautomated synthesis of polypeptides is commercially available fromsuppliers such as Applied Biosystems, Inc., Foster City, Calif. Thus,for example, Leishmania K26, K39 and K9 antigens, or portions thereof,may be synthesized by this method.

Alternatively, the polypeptides of this invention may be prepared byexpression of recombinant DNA encoding the polypeptide in cultured hostcells. Preferably, the host cells are E. coli, yeast, an insect cellline (such as Spodoptera or Trichoplusia) or a mammalian cell line,including (but not limited to) CHO, COS, HEK-293T and NS-1. The DNAsequences expressed in this manner may encode naturally occurringproteins, and fusion proteins comprising Leishmania antigens such asK26, K9 and/or K39, portions thereof, and repeats or other variants ofsuch proteins. Expressed fusion polypeptides of this invention aregenerally isolated in substantially pure form. Preferably, the fusionpolypeptides are isolated to a purity of at least 80% by weight, morepreferably, to a purity of at least 95% by weight, and most preferablyto a purity of at least 99% by weight. In general, such purification maybe achieved using, for example, the standard techniques of ammoniumsulfate fractionation, SDS-PAGE electrophoresis, and affinitychromatography.

Polynucleotide Compositions

The present invention also provides isolated polynucleotides,particularly those encoding the fusion polypeptides of the invention, aswell as compositions comprising such polynucleotides. As used herein,the terms “DNA” and “polynucleotide” and “nucleic acid” refer to a DNAmolecule that has been isolated free of total genomic DNA of aparticular species. Therefore, a DNA segment encoding a polypeptiderefers to a DNA segment that contains one or more coding sequences yetis substantially isolated away from, or purified free from, totalgenomic DNA of the species from which the DNA segment is obtained.Included within the terms “DNA segment” and “polynucleotide” are DNAsegments and smaller fragments of such segments, and also recombinantvectors, including, for example, plasmids, cosmids, phagemids, phage,viruses, and the like.

As will be understood by those skilled in the art, the polynucleotidesequences of this invention can include genomic sequences, extra-genomicand plasmid-encoded sequences and smaller engineered gene segments thatexpress, or may be adapted to express, proteins, fusion polypeptides,peptides and the like. Such segments may be naturally isolated,recombinant, or modified synthetically by the hand of man.

As will be recognized by the skilled artisan, polynucleotides may besingle-stranded (coding or antisense) or double-stranded, and may be DNA(genomic, cDNA or synthetic) or RNA molecules. Additional coding ornon-coding sequences may, but need not, be present within apolynucleotide of the present invention, and a polynucleotide may, butneed not, be linked to other molecules and/or support materials.

Polynucleotides may comprise a native sequence (i.e., an endogenoussequence that encodes a Leishmania antigen or a portion thereof) or maycomprise a variant, or a biological or antigenic functional equivalentof such a sequence. In particular embodiments polynucleotides may encodefor two or more antigenic/immunogenic portions, fragments, or variantsderived from the Leishmania K26, K39, and/or K9 antigens. In certainembodiment, polynucleotides encoding fusion polypeptides of the presentinvention may encode two or more Leishmania antigen fragments as recitedin SEQ ID NOs: 5-7, and 9-11. In a related aspect, the fusionpolypeptide is encoded by polynucleotides encoding the amino acidsequence set forth in SEQ ID NO: 8 or amino acids 10-262 of SEQ ID NO:8.

Polynucleotide variants may contain one or more substitutions,additions, deletions and/or insertions, as further described below,preferably such that the immunogenicity of the encoded polypeptide isnot diminished, relative to the native protein. The effect on theimmunogenicity of the encoded polypeptide may generally be assessed asdescribed herein. The term “variants” also encompasses homologous genesof xenogenic origin.

In additional embodiments, isolated fusion polynucleotides will comprisevarious lengths of contiguous stretches of sequence identical to orcomplementary to two or more K26, K39, and or K9, such as thosesequences disclosed herein, portions or variants thereof. For example,polynucleotides are provided by this invention that comprise at leastabout 15, 20, 30, 40, 50, 75, 100, 150, 200, 300, 400, 500 or 1000 ormore contiguous nucleotides of two or more of the sequences disclosedherein as well as all intermediate lengths there between. It will bereadily understood that “intermediate lengths”, in this context, meansany length between the quoted values, such as 16, 17, 18, 19, etc.; 21,22, 23, etc.; 30, 31, 32, etc.; 50, 51, 52, 53, etc.; 100, 101, 102,103, etc.; 150, 151, 152, 153, etc.; including all integers through200-500; 500-1,000, and the like.

The fusion polynucleotides of the present invention, or fragmentsthereof, regardless of the length of the coding sequence itself, may becombined with other DNA sequences, such as promoters, polyadenylationsignals, additional restriction enzyme sites, multiple cloning sites,other coding segments, and the like, such that their overall length mayvary considerably. It is therefore contemplated that a polynucleotidefragment of almost any length may be employed; with the total lengthpreferably being limited by the ease of preparation and use in theintended recombinant DNA protocol.

Moreover, it will be appreciated by those of ordinary skill in the artthat, as a result of the degeneracy of the genetic code, there are manynucleotide sequences that encode a polypeptide as described herein. Someof these polynucleotides bear minimal homology to the nucleotidesequence of any native gene. Nonetheless, polynucleotides that vary dueto differences in codon usage are specifically contemplated by thepresent invention, for example polynucleotides that are optimized forhuman and/or primate codon selection. Further, alleles of the genescomprising the polynucleotide sequences provided herein are within thescope of the present invention. Alleles are endogenous genes that arealtered as a result of one or more mutations, such as deletions,additions and/or substitutions of nucleotides. The resulting mRNA andprotein may, but need not, have an altered structure or function.Alleles may be identified using standard techniques (such ashybridization, amplification and/or database sequence comparison).

Leishmania polynucleotides and fusions thereof may be prepared,manipulated and/or expressed using any of a variety of well establishedtechniques known and available in the art. For example, polynucleotidesequences or fragments thereof which encode polypeptides of theinvention, or fusion proteins or functional equivalents thereof, may beused in recombinant DNA molecules to direct expression of a polypeptidein appropriate host cells. Due to the inherent degeneracy of the geneticcode, other DNA sequences that encode substantially the same or afunctionally equivalent amino acid sequence may be produced and thesesequences may be used to clone and express a given polypeptide of thepresent invention.

As will be understood by those of skill in the art, it may beadvantageous in some instances to produce fusion polypeptide-encodingnucleotide sequences possessing non-naturally occurring codons. Forexample, codons preferred by a particular prokaryotic or eukaryotic hostcan be selected to increase the rate of protein expression or to producea recombinant RNA transcript having desirable properties, such as ahalf-life which is longer than that of a transcript generated from thenaturally occurring sequence.

Moreover, the polynucleotide sequences of the present invention can beengineered using methods generally known in the art in order to alterfusion polypeptide encoding sequences for a variety of reasons,including but not limited to, alterations which modify the cloning,processing, expression and/or immunogenicity of the gene product.

In order to express a desired fusion polypeptide comprising two or moreantigenic/immunogenic fragments or portions of K26, K39, and/or K9polypeptides, a nucleotide sequence encoding the fusion polypeptide, ora functional equivalent, may be inserted into appropriate expressionvector, i.e., a vector which contains the necessary elements for thetranscription and translation of the inserted coding sequence. Methodswhich are well known to those skilled in the art may be used toconstruct expression vectors containing sequences encoding a polypeptideof interest and appropriate transcriptional and translational controlelements. These methods include in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. Such techniquesare described in Sambrook et al., Molecular Cloning, A Laboratory Manual(2001), and Ausubel et al., Current Protocols in Molecular Biology(January 2008, updated edition).

A variety of expression vector/host systems are known and may beutilized to contain and express polynucleotide sequences. These include,but are not limited to, microorganisms such as bacteria transformed withrecombinant bacteriophage, plasmid, or cosmid DNA expression vectors;yeast transformed with yeast expression vectors; insect cell systemsinfected with virus expression vectors (e.g., baculovirus); plant cellsystems transformed with virus expression vectors (e.g., cauliflowermosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterialexpression vectors (e.g., Ti or pBR322 plasmids); or animal cellsystems.

The “control elements” or “regulatory sequences” present in anexpression vector are those non-translated regions of thevector—enhancers, promoters, 5′ and 3′ untranslated regions—whichinteract with host cellular proteins to carry out transcription andtranslation. Such elements may vary in their strength and specificity.Depending on the vector system and host utilized, any number of suitabletranscription and translation elements, including constitutive andinducible promoters, may be used. For example, when cloning in bacterialsystems, inducible promoters such as the hybrid lacZ promoter of thePBLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) or PSPORT1 plasmid(Gibco BRL, Gaithersburg, Md.) and the like may be used. In mammaliancell systems, promoters from mammalian genes or from mammalian virusesare generally preferred. If it is necessary to generate a cell line thatcontains multiple copies of the sequence encoding a polypeptide, vectorsbased on SV40 or EBV may be advantageously used with an appropriateselectable marker.

In bacterial systems, a number of expression vectors may be selecteddepending upon the use intended for the expressed polypeptide. Forexample, when large quantities are needed, vectors which direct highlevel expression of fusion proteins that are readily purified may beused. Such vectors include, but are not limited to, the multifunctionalE. coli cloning and expression vectors such as PBLUESCRIPT (Stratagene),in which the sequence encoding the polypeptide of interest may beligated into the vector in frame with sequences for the amino-terminalMet and the subsequent 7 residues of β-galactosidase so that a hybridprotein is produced; pIN vectors (Van Heeke & Schuster, J. Biol. Chem.264:5503-5509 (1989)); and the like. PGEX Vectors (Promega, Madison,Wis.) may also be used to express foreign polypeptides as fusionproteins with glutathione S-transferase (GST). In general, such fusionproteins are soluble and can easily be purified from lysed cells byadsorption to glutathione-agarose beads followed by elution in thepresence of free glutathione. Proteins made in such systems may bedesigned to include heparin, thrombin, or factor XA protease cleavagesites so that the cloned polypeptide of interest can be released fromthe GST moiety at will.

In the yeast, Saccharomyces cerevisiae, a number of vectors containingconstitutive or inducible promoters such as alpha factor, alcoholoxidase, and PGH may be used. For reviews, see Ausubel et al. (supra)and Grant et al., Methods Enzymol. 153:516-544 (1987).

In cases where plant expression vectors are used, the expression ofsequences encoding polypeptides may be driven by any of a number ofpromoters. For example, viral promoters such as the 35S and 19Spromoters of CaMV may be used alone or in combination with the omegaleader sequence from TMV (Takamatsu, EMBO J. 6:307-311 (1987)).Alternatively, plant promoters such as the small subunit of RUBISCO orheat shock promoters may be used (Coruzzi et al., EMBO J. 3:1671-1680(1984); Broglie et al., Science 224:838-843 (1984); and Winter et al.,Results Probl. Cell Differ. 17:85-105 (1991)). These constructs can beintroduced into plant cells by direct DNA transformation orpathogen-mediated transfection. Such techniques are described in anumber of generally available reviews (see, e.g., Hobbs in McGraw Hill,Yearbook of Science and Technology, pp. 191-196 (1992)).

An insect system may also be used to express a polypeptide of interest.For example, in one such system, Autographa californica nuclearpolyhedrosis virus (AcNPV) is used as a vector to express foreign genesin Spodoptera frugiperda cells or in Trichoplusia larvae. The sequencesencoding the polypeptide may be cloned into a non-essential region ofthe virus, such as the polyhedrin gene, and placed under control of thepolyhedrin promoter. Successful insertion of the polypeptide-encodingsequence will render the polyhedrin gene inactive and producerecombinant virus lacking coat protein. The recombinant viruses may thenbe used to infect, for example, S. frugiperda cells or Trichoplusialarvae in which the polypeptide of interest may be expressed (Engelhardet al., Proc. Natl. Acad. Sci. U.S.A. 91:3224-3227 (1994)).

In mammalian host cells, a number of viral-based expression systems aregenerally available. For example, in cases where an adenovirus is usedas an expression vector, sequences encoding a polypeptide of the presentinvention may be ligated into an adenovirus transcription/translationcomplex consisting of the late promoter and tripartite leader sequence.Insertion in a non-essential E1 or E3 region of the viral genome may beused to obtain a viable virus which is capable of expressing thepolypeptide in infected host cells (Logan & Shenk, Proc. Natl. Acad.Sci. U.S.A. 81:3655-3659 (1984)). In addition, transcription enhancers,such as the Rous sarcoma virus (RSV) enhancer, may be used to increaseexpression in mammalian host cells.

Specific initiation signals may also be used to achieve more efficienttranslation of sequences encoding a fusion polypeptide of interest. Suchsignals include the ATG initiation codon and adjacent sequences. Incases where sequences encoding the polypeptide, its initiation codon,and upstream sequences are inserted into the appropriate expressionvector, no additional transcriptional or translational control signalsmay be needed. However, in cases where only coding sequence, or aportion thereof, is inserted, exogenous translational control signalsincluding the ATG initiation codon should be provided. Furthermore, theinitiation codon should be in the correct reading frame to ensuretranslation of the entire insert. Exogenous translational elements andinitiation codons may be of various origins, both natural and synthetic.The efficiency of expression may be enhanced by the inclusion ofenhancers which are appropriate for the particular cell system which isused, such as those described in the literature (Scharf. et al., ResultsProbl. Cell Differ. 20:125-162 (1994)).

In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed fusion protein in the desired fashion. Such modifications ofthe polypeptide include, but are not limited to, acetylation,carboxylation, glycosylation, phosphorylation, lipidation, andacylation. Post-translational processing which cleaves a “prepro” formof the protein may also be used to facilitate correct insertion, foldingand/or function. Different host cells such as CHO, HeLa, MDCK, HEK293,and W138, which have specific cellular machinery and characteristicmechanisms for such post-translational activities, may be chosen toensure the correct modification and processing of the foreign protein.

For long-term, high-yield production of recombinant proteins, stableexpression is generally preferred. For example, cell lines which stablyexpress a fusion polynucleotide of the present invention may betransformed using expression vectors which may contain viral origins ofreplication and/or endogenous expression elements and a selectablemarker gene on the same or on a separate vector. Following theintroduction of the vector, cells may be allowed to grow for 1-2 days inan enriched media before they are switched to selective media. Thepurpose of the selectable marker is to confer resistance to selection,and its presence allows growth and recovery of cells which successfullyexpress the introduced sequences. Resistant clones of stably transformedcells may be proliferated using tissue culture techniques appropriate tothe cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase (Wigler et al., Cell 11:223-232 (1977)) and adeninephosphoribosyltransferase (Lowy et al., Cell 22:817-823 (1990)) geneswhich can be employed in tk- or aprt-cells, respectively. Also,antimetabolite, antibiotic or herbicide resistance can be used as thebasis for selection; for example, dhfr which confers resistance tomethotrexate (Wigler et al., Proc. Natl. Acad. Sci. U.S.A. 77:3567-70(1980)); npt, which confers resistance to the aminoglycosides, neomycinand G-418 (Colbere-Garapin et al., J. Mol. Biol. 150:1-14 (1981)); andals or pat, which confer resistance to chlorsulfuron and phosphinotricinacetyltransferase, respectively (Murry, supra). Additional selectablegenes have been described, for example, trpB, which allows cells toutilize indole in place of tryptophan, or hisD, which allows cells toutilize histinol in place of histidine (Hartman & Mulligan, Proc. Natl.Acad. Sci. U.S.A. 85:8047-51 (1988)). The use of visible markers hasgained popularity with such markers as anthocyanins, β-glucuronidase andits substrate GUS, and luciferase and its substrate luciferin, beingwidely used not only to identify transformants, but also to quantify theamount of transient or stable protein expression attributable to aspecific vector system (Rhodes et al., Methods Mol. Biol. 55:121-131(1995)).

A variety of protocols for detecting and measuring the expression ofpolynucleotide-encoded products, using either polyclonal or monoclonalantibodies specific for the product are known in the art. Examplesinclude enzyme-linked immunosorbent assay (ELISA), radioimmunoassay(RIA), and fluorescence activated cell sorting (FACS). These and otherassays are described, among other places, in Hampton et al., SerologicalMethods, a Laboratory Manual (1990) and Maddox et al., J. Exp. Med.158:1211-1216 (1983).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides include oligolabeling,nick translation, end-labeling or PCR amplification using a labelednucleotide. Alternatively, the sequences, or any portions thereof may becloned into a vector for the production of an mRNA probe. Such vectorsare known in the art, are commercially available, and may be used tosynthesize RNA probes in vitro by addition of an appropriate RNApolymerase such as T7, T3, or SP6 and labeled nucleotides. Theseprocedures may be conducted using a variety of commercially availablekits. Suitable reporter molecules or labels, which may be used, includeradionuclides, enzymes, fluorescent, chemiluminescent, or chromogenicagents as well as substrates, cofactors, inhibitors, magnetic particles,and the like.

Host cells transformed with a polynucleotide sequence of interest may becultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The protein produced by a recombinantcell may be secreted or contained intracellularly depending on thesequence and/or the vector used. As will be understood by those of skillin the art, expression vectors containing polynucleotides of theinvention may be designed to contain signal sequences which directsecretion of the encoded polypeptide through a prokaryotic or eukaryoticcell membrane. Other recombinant constructions may be used to joinsequences encoding a polypeptide of interest to nucleotide sequenceencoding a polypeptide domain which will facilitate purification ofsoluble proteins.

In addition to recombinant production methods, fusion polypeptides ofthe invention, and fragments thereof, may be produced by direct peptidesynthesis using solid-phase techniques (Merrifield, J. Am. Chem. Soc.85:2149-2154 (1963)). Protein synthesis may be performed using manualtechniques or by automation. Automated synthesis may be achieved, forexample, using Applied Biosystems 431A Peptide Synthesizer (PerkinElmer). Alternatively, various fragments, for example, two or moreantigenic/immunogenic fragments from Leishmania K26, K39, and/or K9antigens, may be chemically synthesized separately and combined usingchemical methods to produce the full length molecule.

Diagnostic Methods and Kits

In another aspect, this invention provides compounds and methods fordetecting leishmaniasis in individuals and in blood supplies. In aparticular embodiment, the individual is a mammal. In a more particularembodiment the mammal is a human or canine.

In one aspect, there are provided methods for detecting asymptomatic orsub-clinical visceral leishmaniasis in a biological sample, comprising:(a) contacting a biological sample with a fusion polypeptide asdescribed herein, e.g., comprising at least two heterologous Leishmaniaantigens, wherein the Leishmania antigens are selected from K39, K26,and/or K9 or a variant thereof that differs only in conservativesubstitutions and/or modifications; and (b) detecting in the biologicalsample the presence of antibodies that bind to the fusion polypeptide,thereby detecting asymptomatic or sub-clinical visceral Leishmaniainfection in the biological sample.

In a particular aspect, the present invention provides methods fordetecting asymptomatic or sub-clinical visceral leishmaniasis in abiological sample, comprising: (a) contacting a biological sample with afusion polypeptide as described herein, e.g., comprising at least twoheterologous Leishmania antigens, wherein the Leishmania antigens areselected from K39, K26, and/or K9 polypeptide sequences according any ofSEQ ID NOs: 5-7, and 9-11, in any combination, or a variant thereof thatdiffers only in conservative substitutions and/or modifications; and (b)detecting in the biological sample the presence of antibodies that bindto the polypeptide, thereby detecting asymptomatic or sub-clinicalvisceral Leishmania infection in the biological sample. In relatedembodiments, at least one of the Leishmania antigens K26, K39, and K9 isfrom the Leishmania donovani species.

In certain aspects, the present invention provides methods for detectingvisceral Leishmania infection in a biological sample, comprising: (a)contacting a biological sample with a fusion polypeptide as describedherein, e.g., comprising at least two heterologous Leishmania antigens,wherein the fusion protein comprises the amino acid sequence set forthin SEQ ID NO: 8 or amino acids 10-262 of SEQ ID NO: 8, or variantsthereof that differ only in conservative substitutions and/ormodifications; and (b) detecting in the biological sample the presenceof antibodies that bind to the polypeptide, thereby detecting visceralLeishmania infection in the biological sample.

In yet another related aspect, methods are provided for identifying apatient afflicted with asymptomatic or sub-clinical visceralleishmaniasis that is likely to develop acute visceral leishmaniasis. Inone embodiment, the method comprises: (a) contacting a biological sampleobtained from a patient afflicted with asymptomatic or sub-clinicalvisceral leishmaniasis with a fusion polypeptide as described herein,e.g., comprising at least two heterologous Leishmania antigens, whereinthe Leishmania antigens are selected from K39, K26, and/or K9 or avariant thereof that differs only in conservative substitutions and/ormodifications; (b) detecting in the biological sample obtained from apatient the presence of antibodies that bind to the fusion polypeptidein step (a), thereby identifying a patient afflicted with asymptomaticor sub-clinical visceral leishmaniasis that is likely to develop acutevisceral leishmaniasis (VL).

Within related aspects, diagnostic kits for diagnosing leishmaniasis areprovided. In one embodiment, for example, there are provided kits fordetecting visceral leishmaniasis in a biological sample, comprising: (a)a fusion polypeptide as described herein, e.g., comprising at least twoheterologous Leishmania antigens, wherein the Leishmania antigens areselected from K39, K26, and/or K9 or a variant thereof that differs onlyin conservative substitutions and/or modifications; and (b) detecting inthe biological sample the presence of antibodies that bind to thepolypeptide, thereby detecting visceral leishmaniasis in the biologicalsample.

In yet another related aspect, methods are provided for identifying apatient afflicted with asymptomatic or sub-clinical visceralleishmaniasis that is likely to develop acute visceral leishmaniasis. Inone embodiment, the method comprises: (a) contacting a biological sampleobtained from a patient afflicted with asymptomatic or sub-clinicalleishmaniasis with a fusion polypeptide as described herein, e.g.,comprising at least two heterologous Leishmania antigens, wherein theLeishmania antigens are selected from K39, K26, and/or K9 polypeptidesequences according any of SEQ ID NOs: 5-7 and 9-11, in any combination,or a variant thereof that differs only in conservative substitutionsand/or modifications; and (b) detecting in the biological sampleobtained from a patient the presence of antibodies that bind to thefusion polypeptide in step (a), thereby identifying a patient afflictedwith asymptomatic or sub-clinical visceral leishmaniasis that is likelyto develop acute visceral leishmaniasis (VL).

Within related aspects, diagnostic kits for diagnosing leishmaniasis areprovided. In one embodiment, there are provided kits for detectingvisceral leishmaniasis in a biological sample, comprising: (a) a fusionpolypeptide as described herein, e.g., comprising at least twoheterologous Leishmania antigens, wherein the Leishmania antigens areselected from K39, K26, and/or K9 polypeptide sequences according any ofSEQ ID NOs: 5-7 and 9-11, in any combination, or a variant thereof thatdiffers only in conservative substitutions and/or modifications; or avariant thereof that differs only in conservative substitutions and/ormodifications; and (b) detecting in the biological sample the presenceof antibodies that bind to the polypeptide, thereby detecting visceralleishmaniasis in the biological sample.

In yet another related aspect, methods are provided for identifying apatient afflicted with asymptomatic or sub-clinical visceralleishmaniasis that is likely to develop acute visceral leishmaniasis. Inone embodiment, the method comprises: (a) contacting a biological sampleobtained from a patient afflicted with asymptomatic or sub-clinicalleishmaniasis with a fusion polypeptide as described herein, e.g.,comprising at least two heterologous Leishmania antigens, wherein thefusion protein comprises the amino acid sequence set forth in SEQ ID NO:8 or amino acids 10-262 of SEQ ID NO: 8, or variants thereof that differonly in conservative substitutions and/or modifications; and (b)detecting in the biological sample the presence of antibodies that bindto the polypeptide, thereby detecting visceral leishmaniasis in thebiological sample.

Within related aspects, diagnostic kits for diagnosing leishmaniasis areprovided. In one embodiment, there are provided kits for detectingvisceral leishmaniasis in a biological sample, comprising: (a) a fusionpolypeptide as described herein, e.g., comprising at least twoheterologous Leishmania antigens, wherein the fusion protein comprisesthe amino acid sequence set forth in SEQ ID NO: 8 or amino acids 10-262of SEQ ID NO: 8, or variants thereof that differ only in conservativesubstitutions and/or modifications; and (b) detecting in the biologicalsample the presence of antibodies that bind to the polypeptide, therebydetecting visceral leishmaniasis in the biological sample.

In another aspect of this invention, methods are disclosed for detectingand monitoring Leishmania infection, as well as for distinguishing amongtypes of Leishmania infections, in individuals and blood supplies. Ingeneral, Leishmania infection may be detected in any biological samplethat contains antibodies. Preferably, the sample is blood, serum,plasma, saliva, cerebrospinal fluid, stool, or urine. More preferably,the sample is a blood or serum sample obtained from a patient or a bloodsupply.

In another aspect, Leishmania infection may be detected using a fusionpolypeptide comprising two or more polypeptides containing one or moreof the epitopes discussed above, repeats, or variants thereof. Ifmultiple epitopes are employed, these epitopes may be present on one ormore Leishmania antigens. For example, in one aspect, a fusionpolypeptide of the present invention comprises two or more K26, K29,and/or K9 antigenic polypeptides. The fusion polypeptide is then used todetermine the presence or absence of antibodies to the same antigenicpolypeptides in the sample, relative to a predetermined cut-off value.

There are a variety of assay formats known to those of ordinary skill inthe art for using a fusion polypeptide to detect antibodies in a sample.See, e.g., Harlow and Lane, Antibodies. A Laboratory Manual, Cold SpringHarbor Laboratory Press, 1988, which is incorporated herein byreference. In a preferred embodiment, the assay involves the use offusion polypeptide immobilized on a solid support to bind to and removethe antibody from the sample. The bound antibody may then be detectedusing a detection reagent that binds to the antibody/peptide complex andcontains a detectable reporter group. Suitable detection reagentsinclude antibodies that bind to the antibody/polypeptide complex andfree polypeptide labeled with a reporter group (e.g., in asemi-competitive assay). Suitable reporter groups include fluorescentlabels, enzyme labels, radioisotopes, chemiluminescent labels,electrochemiluminescent labels, bioluminescent labels, polymers, polymerparticles, metal particles, haptens, and dyes. Alternatively, acompetitive assay may be utilized, in which an antibody that binds to afusion polypeptide of the present invention labeled with a reportergroup and allowed to bind to the immobilized fusion polypeptide afterincubation of the fusion polypeptide with the sample. The extent towhich components of the sample inhibit the binding of the labeledantibody to the fusion polypeptide is indicative of the reactivity ofthe sample with the immobilized fusion polypeptide.

The solid support may be any material known to those of ordinary skillin the art to which the fusion polypeptide may be attached. For example,the support may be a test well in a microtiter plate or a nitrocelluloseor other suitable membrane. Alternatively, the support may be a bead ordisc, such as glass, fiberglass, latex or a plastic material such aspolystyrene or polyvinylchloride. The support may also be a magneticparticle or a fiber optic sensor, such as those disclosed, for example,in U.S. Pat. No. 5,359,681.

The fusion polypeptide may be bound to the solid support using a varietyof techniques known to those in the art, which are amply described inthe patent and scientific literature. In the context of the presentinvention, the term “bound” refers to both non-covalent association,such as adsorption, and covalent attachment (which may be a directlinkage between the antigen and functional groups on the support or maybe a linkage by way of a cross-linking agent). Binding by adsorption toa well in a microtiter plate or to a membrane is preferred. In suchcases, adsorption may be achieved by contacting the polypeptide, in asuitable buffer, with the solid support for a suitable amount of time.The contact time varies with temperature, but is typically between about1 hour and 1 day. In general, contacting a well of a plastic microtiterplate (such as polystyrene or polyvinylchloride) with an amount offusion polypeptide ranging from about 10 ng to about 1 μg, andpreferably about 100 ng, is sufficient to bind an adequate amount ofantigen. Nitrocellulose will bind approximately 100 μg of protein percm³.

Covalent attachment of fusion polypeptide to a solid support maygenerally be achieved by first reacting the support with a bifunctionalreagent that will react with both the support and a functional group,such as a hydroxyl or amino group, on the fusion polypeptide. Forexample, the fusion polypeptide may be bound to a support having anappropriate polymer coating using benzoquinone or by condensation of analdehyde group on the support with an amine and an active hydrogen onthe polypeptide (see, e.g., Pierce Immunotechnology Catalog and Handbook(1991) at A12-A13).

In certain embodiments, the assay is an enzyme linked immunosorbentassay (ELISA). This assay may be performed by first contacting a fusionpolypeptide of the present invention that has been immobilized on asolid support, commonly the well of a microtiter plate, with the sample,such that antibodies to the Leishmania antigens of the fusionpolypeptide within the sample are allowed to bind to the immobilizedfusion polypeptide. Unbound sample is then removed from the immobilizedfusion polypeptide and a detection reagent capable of binding to theimmobilized antibody-polypeptide complex is added. The amount ofdetection reagent that remains bound to the solid support is thendetermined using a method appropriate for the specific detectionreagent.

Once the fusion polypeptide is immobilized on the support, the remainingprotein binding sites on the support are typically blocked. Any suitableblocking agent known to those of ordinary skill in the art, such asbovine serum albumin (BSA) or Tween 20™ (Sigma Chemical Co., St. Louis,Mo.) may be employed. The immobilized polypeptide is then incubated withthe sample, and antibody (if present in the sample) is allowed to bindto the antigen. The sample may be diluted with a suitable diluent, suchas phosphate-buffered saline (PBS) prior to incubation. In general, anappropriate contact time (i.e., incubation time) is that period of timethat is sufficient to permit detection of the presence of antibodywithin a Leishmania-infected sample. Preferably, the contact time issufficient to achieve a level of binding that is at least 95% of thatachieved at equilibrium between bound and unbound antibody. Those ofordinary skill in the art will recognize that the time necessary toachieve equilibrium may be readily determined by assaying the level ofbinding that occurs over a period of time. At room temperature, anincubation time of about 30 minutes is generally sufficient.

Unbound sample may then be removed by washing the solid support with anappropriate buffer, such as PBS containing 0.1% Tween 20™. Detectionreagent may then be added to the solid support. An appropriate detectionreagent is any compound that binds to the immobilizedantibody-polypeptide complex and that can be detected by any of avariety of means known to those in the art. Preferably, the detectionreagent contains a binding agent (such as, for example, Protein A,Protein G, immunoglobulin, lectin or free antigen) conjugated to areporter group. Preferred reporter groups include enzymes (such ashorseradish peroxidase), substrates, cofactors, inhibitors, dyes,radionuclides, luminescent groups, fluorescent groups and biotin. Theconjugation of binding agent to reporter group may be achieved usingstandard methods known to those of ordinary skill in the art. Commonbinding agents may also be purchased conjugated to a variety of reportergroups from many sources (e.g., Zymed Laboratories, San Francisco,Calif. and Pierce, Rockford, Ill.).

The detection reagent is then incubated with the immobilized antibodypolypeptide complex for an amount of time sufficient to detect the boundantibody. An appropriate amount of time may generally be determined fromthe manufacturer's instructions or by assaying the level of binding thatoccurs over a period of time. Unbound detection reagent is then removedand bound detection reagent is detected using the reporter group. Themethod employed for detecting the reporter group depends upon the natureof the reporter group. For radioactive groups, scintillation counting orautoradiographic methods are generally appropriate. Pectroscopic methodsmay be used to detect dyes, luminescent groups and fluorescent groups.Biotin may be detected using avidin, coupled to a different reportergroup (commonly a radioactive or fluorescent group or an enzyme). Enzymereporter groups may generally be detected by the addition of substrate(generally for a specific period of time), followed by spectroscopic orother analysis of the reaction products.

To determine the presence or absence of anti-Leishmania antibodies inthe sample, the signal detected from the reporter group that remainsbound to the solid support is generally compared to a signal thatcorresponds to a predetermined cut-off value. In one preferredembodiment, the cut-off value is preferably the average mean signalobtained when the immobilized polypeptide is incubated with samples froman uninfected patient. In general, a sample generating a signal that isthree standard deviations above the predetermined cut-off value isconsidered positive (i.e., reactive with the polypeptide). In analternate preferred embodiment, the cut-off value is determined using aReceiver Operator Curve, according to the method of Sackett et al.,Clinical Epidemiology: A Basic Science for Clinical Medicine, p. 106-7(Little Brown and Co., 1985). Briefly, in this embodiment, the cut-offvalue may be determined from a plot of pairs of true positive rates(i.e., sensitivity) and false positive rates (100%-specificity) thatcorrespond to each possible cut-off value for the diagnostic testresult. The cut-off value on the plot that is the closest to the upperlefthand corner (i.e., the value that encloses the largest area) is themost accurate cut-off value, and a sample generating a signal that ishigher than the cut-off value determined by this method may beconsidered positive. Alternatively, the cut-off value may be shifted tothe left along the plot, to minimize the false positive rate, or to theright, to minimize the false negative rate.

In a related embodiment, the assay is performed in a flow-through orstrip test format, wherein the antigen is immobilized on a membrane suchas nitrocellulose. In the flow-through test, antibodies within thesample bind to the immobilized polypeptide as the sample passes throughthe membrane. A detection reagent (e.g., protein A-colloidal gold) thenbinds to the antibody-polypeptide complex as the solution containing thedetection reagent flows through the membrane. The detection of bounddetection reagent may then be performed as described above. In the striptest format, one end of the membrane to which polypeptide is bound isimmersed in a solution containing the sample. The sample migrates alongthe membrane through a region containing detection reagent and to thearea of immobilized fusion polypeptide. Concentration of detectionreagent at the fusion polypeptide indicates the presence of Leishmaniaantibodies in the sample. Typically, the concentration of detectionreagent at that site generates a pattern, such as a line, that can beread visually. The absence of such a pattern indicates a negativeresult. In general, the amount of fusion polypeptide immobilized on themembrane is selected to generate a visually discernible pattern when thebiological sample contains a level of antibodies that would besufficient to generate a positive signal in an ELISA, as discussedabove. Preferably, the amount of fusion polypeptide immobilized on themembrane ranges from about 25 ng to about 1 μg, and more preferably fromabout 50 ng to about 500 ng. Such tests can typically be performed witha very small amount (e.g., one drop) of patient serum or blood.

Of course, numerous other assay protocols exist that are suitable foruse with the fusion polypeptides of the present invention. The abovedescriptions are intended to be exemplary only.

In one aspect of the invention, the assays discussed above may be usedto specifically detect visceral leishmaniasis. In this aspect,antibodies in the sample may be detected using a fusion polypeptidecomprising the amino acid sequence of two or more antigenic/immunogenicfragments or epitopes of a Leishmania K26, K39, and/or K9 antigen. Inanother aspect, antibodies in the sample may be detected using a fusionpolypeptide comprising the amino acid sequence of two or moreimmunogenic fragments or epitopes as set forth in any of SEQ ID NOs: 5-7and 9-11. In another aspect, antibodies in the sample may be detectedusing a fusion polypeptide comprising the amino acid sequence set forthin SEQ ID NO: 8 or amino acids 10-262 of SEQ ID NO: 8. Preferably, theLeishmania antigens are immobilized by adsorption to a solid supportsuch as a well of a microtiter plate or a membrane, as described above,in roughly similar amounts such that the total amount of fusionpolypeptide in contact with the support ranges from about 10 ng to about100 μg. The remainder of the steps in the assay may generally beperformed as described above. It will be readily apparent to those ofordinary skill in the art that, by combining polypeptides describedherein with other polypeptides that can detect cutaneous and mucosalleishmaniasis, the polypeptides disclosed herein may be used in methodsthat detect all types of leishmaniasis.

In another aspect of the invention, patients with asymptomatic orsub-clinical VL whose disease is likely to progress to acute visceralleishmaniasis may be distinguished from infected patients whose diseaseis not likely to progress. Such progression may occur within a year (andtypically within 5-12 months) for sub-clinical disease, or within manyyears in the case of asymptomatic patients. This determination may bemade using any of several approaches. In one embodiment, the assay isperformed using a polypeptide as described herein, e.g., that comprisesat least one repeat unit of the K39 antigen, as set forth in SEQ ID NOs:6, 10 or 11, for example. In a related embodiment, the polypeptidecomprises a K39 repeat unit antigen encoded by the polynucleotidesequence recited in SEQ ID NOs: 2, 14 or 15. While a K39 repeat unitantigen generates a positive result (relative to the predeterminedcut-off value) when reacted with sera from more than 97% of patientswith acute visceral leishmaniasis, patients with asymptomaticleishmaniasis react very weakly, if at all, with this antigen. Thosesera that react weakly are likely to indicate infections that are in theprocess of progression, or are likely to progress, to acute visceralleishmaniasis (or infections that are in remission or responding totreatment, which may be distinguished based on patient history).

In another embodiment, the assay is separately performed with a fusionpolypeptide of the invention, e.g., comprising the amino acid sequenceof two or more antigenic/immunogenic fragments or epitopes of aLeishmania K26, K39, and/or K9 antigen, such as K28 for example, andwith a polypeptide that comprises at least one repeat unit of the K39antigen. In this embodiment, the optical density (OD) obtained in theassay using the K28 fusion polypeptide is compared to the value obtainedusing the K39 polypeptide. A significantly higher OD in the assay usingthe K28 fusion polypeptide, when compared to the OD in the assay usingthe K39 polypeptide indicates a more robust, reliable detection of anasymptomatic or sub-clinical VL infection. Those asymptomatic orsub-clinical patients for whom both values are relatively high arelikely to be in the process of developing acute visceral leishmaniasis(or in the process of recovering from infection). In another aspect, theassay is separately performed with a fusion polypeptide comprising theamino acid sequence of two or more immunogenic fragments or epitopes asset forth in any of SEQ ID NOs: 5-7 and 9-11 and with a polypeptide thatcomprises at least one repeat unit of a K39 antigen. In another aspect,the assay is separately performed with a fusion polypeptide comprisingthe amino acid sequence set forth in SEQ ID NO: 8 or amino acids 10-262of SEQ ID NO: 8, and with a polypeptide that comprises at least onerepeat unit of a K39 antigen.

In another embodiment, asymptomatic or sub-clinical patients that arelikely to develop acute visceral leishmaniasis may be identified usingseparate fusion polypeptide (e.g., K28) and K39 polypeptide assays (asdescribed above) that are performed over a period of time. For example,the assays may be performed every 1, 2, 3, 4, 5, or 6 months for aperiod of months or years. Asymptomatic or sub-clinical patients thatare likely to remain asymptomatic or sub-clinical will generally havesera that show a high reactivity with K28 and a low reactivity with theK39 polypeptide, as discussed above, at each time point. However,patients that are progressing toward acute visceral leishmaniasis willshow an increase in the reactivity of both K28 and K39 polypeptides overthe time period of the assays. By monitoring an individual patient inthis manner, the development of acute visceral leishmaniasis may beidentified before other symptoms become apparent. This earlyidentification allows selective treatment of only those asymptomaticpatients that are predisposed to develop a more serious form of thedisease.

In another aspect of this invention, immobilized fusion polypeptides maybe used to purify antibodies that bind thereto. Such antibodies may beprepared by any of a variety of techniques known to those of ordinaryskill in the art. See, e.g., Harlow and Land, Antibodies. A LaboratoryManual, Cold Spring Harbor Laboratory Press, 1988. In one suchtechnique, an immunogen comprising a fusion polypeptide of the presentinvention is initially injected into any of a wide variety of mammals(e.g., mice, rats, rabbits, sheep and goats). In this step, thepolypeptide may serve as the immunogen without modification.Alternatively, particularly for relatively short polypeptides, asuperior immune response may be elicited if the polypeptide is joined toa carrier protein, such as bovine serum albumin or keyhole limpethemocyanin. The immunogen is injected into the animal host, preferablyaccording to a predetermined schedule incorporating one or more boosterimmunizations, and the animals are bled periodically. Polyclonalantibodies specific for the polypeptide may then be purified from suchantisera by, for example, affinity chromatography using the polypeptidecoupled to a suitable solid support.

Monoclonal antibodies specific for the antigenic fusion polypeptide ofinterest may be prepared, for example, using the technique of Kohler andMilstein, Eur. J. Immunol. 6:511-519, 1976, and improvements thereto.Briefly, these methods involve the preparation of immortal cell linescapable of producing antibodies having the desired specificity (i.e.,reactivity with the polypeptide of interest). Such cell lines may beproduced, for example, from spleen cells obtained from an animalimmunized as described above. The spleen cells are then immortalized by,for example, fusion with a myeloma cell fusion partner, preferably onethat is syngeneic with the immunized animal. A variety of fusiontechniques may be employed. For example, the spleen cells and myelomacells may be combined with a nonionic detergent for a few minutes andthen plated at low density on a selective medium that supports thegrowth of hybrid cells, but not myeloma cells. A preferred selectiontechnique uses HAT (hypoxanthine, aminopterin, thymidine) selection.After a sufficient time, usually about 1 to 2 weeks, colonies of hybridsare observed. Single colonies are selected and tested for bindingactivity against the polypeptide. Hybridomas having high reactivity andspecificity are preferred.

Monoclonal antibodies may be isolated from the supernatants of growinghybridoma colonies. In this process, various techniques may be employedto enhance the yield, such as injection of the hybridoma cell line intothe peritoneal cavity of a suitable vertebrate host, such as a mouse.Monoclonal antibodies may then be harvested from the ascites fluid orthe blood. Contaminants may be removed from the antibodies byconventional techniques, such as chromatography, gel filtration,precipitation, and extraction. One or more polypeptides may be used inthe purification process in, for example, an affinity chromatographystep.

Monospecific antibodies that bind to a fusion polypeptide comprising twoor more immunogenic portions of Leishmania K26, K39, and/or K9 antigensmay be used, for example, to detect Leishmania infection in a biologicalsample using one of a variety of immunoassays, which may be direct orcompetitive. Briefly, in one direct assay format, a monospecificantibody may be immobilized on a solid support (as described above) andcontacted with the sample to be tested. After removal of the unboundsample, a second monospecific antibody, which has been labeled with areporter group, may be added and used to detect bound antigen. In anexemplary competitive assay, the sample may be combined with themonoclonal or polyclonal antibody, which has been labeled with asuitable reporter group. The mixture of sample and antibody may then becombined with polypeptide antigen immobilized on a suitable solidsupport. Antibody that has not bound to an antigen in the sample isallowed to bind to the immobilized antigen and the remainder of thesample and antibody is removed. The level of antibody bound to the solidsupport is inversely related to the level of antigen in the sample.Thus, a lower level of antibody bound to the solid support indicates thepresence of Leishmania in the sample. Other formats for usingmonospecific antibodies to detect Leishmania in a sample will beapparent to those of ordinary skill in the art, and the above formatsare provided solely for exemplary purposes.

Pharmaceutical and Vaccine Compositions

In another aspect, the present invention concerns formulations of one ormore of the polynucleotide, fusion polypeptide or other compositionsdisclosed herein in pharmaceutically-acceptable orphysiologically-acceptable solutions for administration to a cell or ananimal, either alone, or in combination with one or more othermodalities of therapy. Such pharmaceutical compositions are particularlypreferred for use as vaccines when formulated with a suitableimmunostimulant/adjuvant system. The compositions are also suitable foruse in a diagnostic context.

It will also be understood that, if desired, the compositions of theinvention may be administered in combination with other agents as well,such as, e.g., other proteins or polypeptides or variouspharmaceutically-active agents. There is virtually no limit to othercomponents that may also be included, provided that the additionalagents do not cause a significant adverse effect upon the objectivesaccording to the invention.

In certain embodiments, the compositions of the invention are used asvaccines and are formulated in combination with one or moreimmunostimulants. An immunostimulant may be any substance that enhancesor potentiates an immune response (antibody and/or cell-mediated) to anexogenous antigen. Examples of immunostimulants include adjuvants,biodegradable microspheres (e.g., polylactic galactide) and liposomes(into which the compound is incorporated; see, e.g., Fullerton, U.S.Pat. No. 4,235,877). Vaccine preparation is generally described in, forexample, Powell & Newman, eds., Vaccine Design (the subunit and adjuvantapproach) (1995).

Any of a variety of immunostimulants may be employed in the vaccines ofthis invention. For example, an adjuvant may be included. Many adjuvantscontain a substance designed to protect the antigen from rapidcatabolism, such as aluminum hydroxide or mineral oil, and a stimulatorof immune responses, such as lipid A (natural or synthetic), Bortadellapertussis or Mycobacterium species or Mycobacterium derived proteins.Suitable adjuvants are commercially available as, for example, Freund'sIncomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit,Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.); AS-2and derivatives thereof (SmithKline Beecham, Philadelphia, Pa.); CWS,TDM, Leif, aluminum salts such as aluminum hydroxide gel (alum) oraluminum phosphate; salts of calcium, iron or zinc; an insolublesuspension of acylated tyrosine; acylated sugars; cationically oranionically derivatized polysaccharides; polyphosphazenes; biodegradablemicrospheres; monophosphoryl lipid A and quil A. Cytokines, such asGM-CSF or interleukin-2, -7, or -12, may also be used as adjuvants.

Other illustrative adjuvants useful in the context of the inventioninclude Toll-like receptor agonists, such as TLR7 agonists, TLR7/8agonists, and the like. Still other illustrative adjuvants includeimiquimod, gardiquimod, resiquimod, and related compounds.

Certain vaccines employ adjuvant systems designed to induce an immuneresponse predominantly of the Th1 type. High levels of Th1-typecytokines (e.g., IFN-γ, TNF-α, IL-2 and IL-12) tend to favor theinduction of cell mediated immune responses to an administered antigen.In contrast, high levels of Th2-type cytokines (e.g., IL-4, IL-5, IL-6and IL-10) tend to favor the induction of humoral immune responses.Following application of a vaccine as provided herein, a patient willsupport an immune response that includes Th1- and Th2-type responses.Within one embodiment, in which a response is predominantly Th1-type,the level of Th1-type cytokines will increase to a greater extent thanthe level of Th2-type cytokines. The levels of these cytokines may bereadily assessed using standard assays. For a review of the families ofcytokines, see Mossman & Coffman, Ann. Rev. Immunol. 7:145-173 (1989).

Certain adjuvants for use in eliciting a predominantly Th1-type responseinclude, for example, a combination of monophosphoryl lipid A,preferably 3-de-O-acylated monophosphoryl lipid A (3D-MPL™), togetherwith an aluminum salt (U.S. Pat. Nos. 4,436,727; 4,877,611; 4,866,034;and 4,912,094). CpG-containing oligonucleotides (in which the CpGdinucleotide is unmethylated) also induce a predominantly Th1 response.Such oligonucleotides are well known and are described, for example, inWO 96/02555, WO 99/33488 and U.S. Pat. Nos. 6,008,200 and 5,856,462.Immunostimulatory DNA sequences are also described, for example, by Satoet al., Science 273:352 (1996). Another illustrative adjuvant comprisesa saponin, such as Quil A, or derivatives thereof, including QS21 andQS7 (Aquila Biopharmaceuticals Inc., Framingham, Mass.); Escin;Digitonin; or Gypsophila or Chenopodium quinoa saponins. Otherillustrative formulations include more than one saponin in the adjuvantcombinations of the present invention, for example combinations of atleast two of the following group comprising QS21, QS7, Quil A, escin, ordigitonin.

In a particular embodiment, the adjuvant system includes the combinationof a monophosphoryl lipid A and a saponin derivative, such as thecombination of QS21 and 3D-MPL™. adjuvant, as described in WO 94/00153,or a less reactogenic composition where the QS21 is quenched withcholesterol, as described in WO 96/33739. Other formulations comprise anoil-in-water emulsion and tocopherol. Another adjuvant formulationemploying QS21, 3D-MPL™ adjuvant and tocopherol in an oil-in-wateremulsion is described in WO 95/17210.

Another enhanced adjuvant system involves the combination of aCpG-containing oligonucleotide and a saponin derivative as disclosed inWO 00/09159.

Other illustrative adjuvants include Montanide ISA 720 (Seppic, France),SAF (Chiron, Calif., United States), ISCOMS (CSL), MF-59 (Chiron), theSBAS series of adjuvants (e.g., SBAS-2, AS2′, AS2,″ SBAS-4, or SBAS6,available from SmithKline Beecham, Rixensart, Belgium), Detox, RC-529(Corixa, Hamilton, Mont.) and other aminoalkyl glucosaminide4-phosphates (AGPs), such as those described in pending U.S. patentapplication Ser. Nos. 08/853,826 and 09/074,720, the disclosures ofwhich are incorporated herein by reference in their entireties, andpolyoxyethylene ether adjuvants such as those described in WO99/52549A1.

Compositions of the invention may also, or alternatively, comprise Tcells specific for a Leishmania fusion polypeptide as described hereinthroughout. Such cells may generally be prepared in vitro or ex vivo,using standard procedures. For example, T cells may be isolated frombone marrow, peripheral blood, or a fraction of bone marrow orperipheral blood of a patient. Alternatively, T cells may be derivedfrom related or unrelated humans, non-human mammals, cell lines orcultures.

T cells may be stimulated with a fusion polypeptide comprising, two ormore immunogenic portions of Leishmania K26, K39, and/or K9 antigens,polynucleotide encoding such a fusion polypeptide, and/or an antigenpresenting cell (APC) that expresses such a fusion polypeptide. Suchstimulation is performed under conditions and for a time sufficient topermit the generation of T cells that are specific for the fusionpolypeptide. Preferably, the fusion polypeptide or polynucleotide ispresent within a delivery vehicle, such as a microsphere, to facilitatethe generation of specific T cells.

T cells are considered to be specific for a fusion polypeptide of theinvention if the T cells specifically proliferate, secrete cytokines orkill target cells coated with the polypeptide or expressing a geneencoding the polypeptide. T cell specificity may be evaluated using anyof a variety of standard techniques. For example, within a chromiumrelease assay or proliferation assay, a stimulation index of more thantwo fold increase in lysis and/or proliferation, compared to negativecontrols, indicates T cell specificity. Such assays may be performed,for example, as described in Chen et al., Cancer Res. 54:1065-1070(1994)). Alternatively, detection of the proliferation of T cells may beaccomplished by a variety of known techniques. For example, T cellproliferation can be detected by measuring an increased rate of DNAsynthesis (e.g., by pulse-labeling cultures of T cells with tritiatedthymidine and measuring the amount of tritiated thymidine incorporatedinto DNA). Contact with a polypeptide of the invention (100 ng/ml-100μg/ml, preferably 200 ng/ml-25 μg/ml) for 3-7 days should result in atleast a two fold increase in proliferation of the T cells. Contact asdescribed above for 2-3 hours should result in activation of the Tcells, as measured using standard cytokine assays in which a two foldincrease in the level of cytokine release (e.g., TNF-α or IFN-γ) isindicative of T cell activation (see Coligan et al., Current Protocolsin Immunology, vol. 1 (1998)). T cells that have been activated inresponse to a fusion polypeptide, polynucleotide or fusionpolypeptide-expressing APC may be CD4+ and/or CD8+. Protein-specific Tcells may be expanded using standard techniques. Within preferredembodiments, the T cells are derived from a patient, a related donor oran unrelated donor, and are administered to the patient followingstimulation and expansion.

In the pharmaceutical compositions of the invention, formulation ofpharmaceutically-acceptable excipients and carrier solutions iswell-known to those of skill in the art, as is the development ofsuitable dosing and treatment regimens for using the particularcompositions described herein in a variety of treatment regimens,including e.g., oral, parenteral, intravenous, intranasal, andintramuscular administration and formulation.

In certain applications, the pharmaceutical compositions disclosedherein may be delivered via oral administration to a subject. As such,these compositions may be formulated with an inert diluent or with anassimilable edible carrier, or they may be enclosed in hard- orsoft-shell gelatin capsule, or they may be compressed into tablets, orthey may be incorporated directly with the food of the diet.

In certain circumstances it will be desirable to deliver thepharmaceutical compositions disclosed herein parenterally,intravenously, intramuscularly, or even intraperitoneally as described,for example, in U.S. Pat. No. 5,543,158; U.S. Pat. No. 5,641,515 andU.S. Pat. No. 5,399,363 (each specifically incorporated herein byreference in its entirety). Solutions of the active compounds as freebase or pharmacologically acceptable salts may be prepared in watersuitably mixed with a surfactant, such as hydroxypropylcellulose.Dispersions may also be prepared in glycerol, liquid polyethyleneglycols, and mixtures thereof and in oils. Under ordinary conditions ofstorage and use, these preparations contain a preservative to preventthe growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions (U.S. Pat. No. 5,466,468, specifically incorporated hereinby reference in its entirety). In all cases the form must be sterile andmust be fluid to the extent that easy syringability exists. It must bestable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms, such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (e.g., glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and/or vegetable oils. Proper fluidity may bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. The prevention of the action ofmicroorganisms can be facilitated by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, a sterile aqueous medium that can be employed will be knownto those of skill in the art in light of the present disclosure. Forexample, one dosage may be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion (see, e.g., Remington's PharmaceuticalSciences, 15th Edition, pp. 1035-1038 and 1570-1580). Some variation indosage will necessarily occur depending on the condition of the subjectbeing treated. The person responsible for administration will, in anyevent, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, and the general safety and purity standards as required byFDA Office of Biologics standards.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with thevarious other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

The compositions disclosed herein may be formulated in a neutral or saltform. Pharmaceutically-acceptable salts include the acid addition salts(formed with the free amino groups of the protein) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, oxalic, tartaric, mandelic, andthe like. Salts formed with the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, histidine, procaine and the like. Upon formulation,solutions will be administered in a manner compatible with the dosageformulation and in such amount as is therapeutically effective. Theformulations are easily administered in a variety of dosage forms suchas injectable solutions, drug-release capsules, and the like.

As used herein, “carrier” includes any and all solvents, dispersionmedia, vehicles, coatings, diluents, antibacterial and antifungalagents, isotonic and absorption delaying agents, buffers, carriersolutions, suspensions, colloids, and the like. The use of such mediaand agents for pharmaceutical active substances is well known in theart. Except insofar as any conventional media or agent is incompatiblewith the active ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients can also be incorporatedinto the compositions.

The phrase “pharmaceutically-acceptable” refers to molecular entitiesand compositions that do not produce an allergic or similar untowardreaction when administered to a human. The preparation of an aqueouscomposition that contains a protein as an active ingredient is wellunderstood in the art. Typically, such compositions are prepared asinjectables, either as liquid solutions or suspensions; solid formssuitable for solution in, or suspension in, liquid prior to injectioncan also be prepared. The preparation can also be emulsified.

In certain embodiments, the pharmaceutical compositions may be deliveredby intranasal sprays, inhalation, and/or other aerosol deliveryvehicles. Methods for delivering genes, polynucleotides, and peptidecompositions directly to the lungs via nasal aerosol sprays has beendescribed e.g., in U.S. Pat. No. 5,756,353 and U.S. Pat. No. 5,804,212(each specifically incorporated herein by reference in its entirety).Likewise, the delivery of drugs using intranasal microparticle resins(Takenaga et al., 1998) and lysophosphatidyl-glycerol compounds (U.S.Pat. No. 5,725,871, specifically incorporated herein by reference in itsentirety) are also well-known in the pharmaceutical arts. Likewise,transmucosal drug delivery in the form of a polytetrafluoroetheylenesupport matrix is described in U.S. Pat. No. 5,780,045 (specificallyincorporated herein by reference in its entirety).

In certain embodiments, the delivery may occur by use of liposomes,nanocapsules, microparticles, microspheres, lipid particles, vesicles,and the like, for the introduction of the compositions of the presentinvention into suitable host cells. In particular, the compositions ofthe present invention may be formulated for delivery either encapsulatedin a lipid particle, a liposome, a vesicle, a nanosphere, a nanoparticleor the like. The formulation and use of such delivery vehicles can becarried out using known and conventional techniques.

In another aspect of this invention, vaccines and pharmaceuticalcompositions are provided for the prevention of Leishmania infection,and complications thereof, in a mammal, preferably a human or dog.Pharmaceutical compositions generally comprise one or more fusionpolypeptides as described herein, and a physiologically acceptablecarrier. The vaccines comprise one or more of the above fusionpolypeptides and an adjuvant, for enhancement of the immune response.

Routes and frequency of administration and fusion polypeptide doses willvary from individual to individual and may parallel those currentlybeing used in immunization against other protozoan infections. Ingeneral, the pharmaceutical compositions and vaccines may beadministered by injection (e.g., intramuscular, intravenous orsubcutaneous), intranasally (e.g., by aspiration) or orally. Between 1and 4 doses may be administered for a 2-6 week period. Preferably, twodoses are administered, with the second dose 2-4 weeks later than thefirst. A suitable dose is an amount of fusion polypeptide that iseffective to raise antibodies in a treated mammal that are sufficient toprotect the mammal from Leishmania infection for a period of time. Ingeneral, the amount of fusion polypeptide present in a dose ranges fromabout 1 pg to about 100 mg per kg of host, typically from about 10 pg toabout 1 mg, and preferably from about 100 pg to about 1 μg. Suitabledose sizes will vary with the size of the animal, but will typicallyrange from about 0.01 mL to about 5 mL for 10-60 kg animal.

While any suitable carrier known to those of ordinary skill in the artmay be employed in the pharmaceutical compositions of this invention,the type of carrier will vary depending on the mode of administration.For parenteral administration, such as subcutaneous injection, thecarrier preferably comprises water, saline, alcohol, a fat, a wax or abuffer. For oral administration, any of the above carriers or a solidcarrier, such as mannitol, lactose, starch, magnesium stearate, sodiumsaccharine, talcum, cellulose, glucose, sucrose, and magnesiumcarbonate, may be employed. Biodegradable microspheres (e.g., polylacticgalactide) may also be employed as carriers for the pharmaceuticalcompositions of this invention.

Any of a variety of adjuvants may be employed in the vaccines of thisinvention to nonspecifically enhance the immune response. Most adjuvantscontain a substance designed to protect the antigen from rapidcatabolism, such as aluminum hydroxide or mineral oil, and a nonspecificstimulator of immune response, such as lipid A, Bordella pertussis orMycobacterium tuberculosis. Such adjuvants are commercially availableas, for example, Freund's Incomplete Adjuvant and Complete Adjuvant(Difco Laboratories, Detroit, Mich.) and Merck Adjuvant 65 (Merck andCompany, Inc., Rahway, N.J.).

The various embodiments described above can be combined to providefurther embodiments. All of the U.S. patents, U.S. patent applicationpublications, U.S. patent applications, foreign patents, foreign patentapplications and non-patent publications referred to in thisspecification and/or listed in the Application Data Sheet, areincorporated herein by reference, in their entirety. Aspects of theembodiments can be modified, if necessary to employ concepts of thevarious patents, applications and publications to provide yet furtherembodiments.

EXAMPLES Example 1 Cloning and Expression of Leishmania FusionPolypeptide

The present invention relates to a synthetic gene construct, referred toas K28, comprising sequences derived from 3 Leishmania donovani genes.The synthetic gene comprises partial DNA sequences of the K26 and K39genes and the complete DNA sequences of the K9 gene. Additionally, thesynthetic gene comprises a nine amino acid N-terminal motif, whichincludes 6 histidines (e.g., a 6× HIS epitope tag). The synthetic DNAconstruct was cloned into the NdeI/XhoI site of plasmid pCRX2.1 in orderto express the fusion protein of this gene. The synthetic gene containsthree 14 amino acid repeats of K26 encoded by SEQ ID NO: 1, two 39 aminoacid repeats of K39 encoded by SEQ ID NO: 2, and the complete openreading frame for the K9 gene encoded by SEQ ID NO: 3. This fusionconstruct was designed to improve the diagnostic potential of each ofthese single proteins into one molecule and offers a broader coverage,increased sensitivity and reduced costs in terms of manufacturing asingle protein instead of all three.

Example 2 Detection of Asymptomatic and Sub-Clinical Visceral LeishmaniaInfections in Humans Using a K28 Polypeptide

This Example illustrates the increased detection sensitivity ofLeishmania infection in humans using a fusion polypeptide of theinvention, prepared as described in Example 1, in an ELISA format.

The ELISA assays were performed as follows. Three series of Polysorp 96well plates (Nunc, Rochester, N.Y.) were each coated with 2 μg/ml of adifferent recombinant antigen in bicarbonate buffer overnight at 4° C.and blocked for 2 hours at room temperature with PBST with 1% (w/v) BSAon a plate shaker. Sera were diluted appropriately to 1/200 in PBST with0.1% BSA, added to each well and plates were incubated at roomtemperature for 2 hours with shaking. Plates were washed with PBST with0.1% BSA and then HRP conjugated IgG immunoglobulin (Sigma, St. Louis,Mo.), diluted 1:10000 in PBST and 0.1% BSA, was added to each well andincubated at room temperature for 60 minutes with shaking. Afterwashing, plates were developed with peroxidase color substrate (KPL,Baltimore Md.) with reaction quenched by addition of 1N H₂SO₄ after 10minutes. The corrected optical density of each well at 450-570 nm wasread using a VERSAmax® microplate reader (Molecular Devices, Sunnyvale,Calif.). The cut-off value was determined for each test by calculatingthe mean of the Enzyme Conjugate negative controls plus three standarddeviations (EC).

Venezuelan individuals with visceral leishmaniasis (VL) were identifiedbased on serology (e.g., IFAT or IHA immunofluorescence orhemaglutination), clinical symptoms (e.g., malaise, diarrhea,splenomegaly and hepatomegaly) and whole lysate ELISA. Of 52 serumsamples from patients with VL, 94% tested positive using the aboveassay. However, the K28 antigen test displayed significantly higher ODsin 33% of the weak or marginally detected samples using the K39 antigenalone. In addition, 3 samples that were initially characterized asnegative using the K39 assay were reliably detected using the K28antigen test.

These results are depicted in FIG. 2, which show the distribution ofabsorbance values at 450-570 nm for the 52 VL serum samples assayed withK26, K28, and K39. These results indicate that using K28 providesincreased sensitivity over using K39 alone to detect asymptomatic orsub-clinical visceral leishmaniasis.

Example 3 Detection of Asymptomatic and Sub-Clinical Visceral LeishmaniaInfections in Canines Using a K28 Polypeptide

This Example illustrates the increased detection sensitivity ofLeishmania infection in canines using a fusion polypeptide of theinvention, prepared as described in Example 1, in an ELISA format.

The ELISA assays were performed as described in Example 2, except that 4different recombinant antigens were used. Four series of Polysorp 96well plates were each coated with 2 μg/ml of either K9, K26, K28, or K39recombinant antigens. Venezuelan canines with visceral leishmaniasis(VL) were identified based on serology (e.g., IFAT or IHAimmunofluorescence or hemaglutination), clinical symptoms (e.g.,malaise, diarrhea, splenomegaly and hepatomegaly) and whole lysateELISA.

The results depicted in FIG. 3 show that K28 identified a significantlygreater number of cases than either of the other three antigens usedalone. This indicates that using K28 provides increased sensitivity incanines to detect asymptomatic or sub-clinical leishmaniasis, andmoreover, K28 provides increased sensitivity of detection over using K9,K26, and K39 antigens alone.

Example 4 Detection of Visceral Leishmania Infections in Humans Using aK28 Polypeptide or a K39 Polypeptide in a Dual Path Rapid Test FormatCompared to Direct Agllutination Assay

This Example illustrates the increased detection sensitivity ofLeishmania infection in humans using a fusion polypeptide of theinvention (K28), prepared as described in Example 1, versus a singleantigen (K39) in a dual path rapid test format, as compared to a directagglutination assay (DAT) using sera from Sudan.

The dual path rapid test was performed essentially as described in U.S.Pat. No. 7,189,522, which is herein incorporated by reference in itsentirety. Briefly, K28 or K39 was coated onto a strip of nitrocellulose.A drop of appropriately diluted human serum was added to the sample wellin a test cartridge containing the antigen-impregnated nitrocellulose.Two drops of sample buffer were then added to the sample well and thesample was allowed to migrate by capillary action until the lines in thedetection window of the test cartridge disappeared. Two drops of bufferwere then added to the detection reagent well in the test cartridge toreconstitute immobilized detection reagent (colloidal gold-conjugatedStaphylococcal protein A).

The detection reagent migrates by capillary flow to the detectionwindow. If the sample contains antibodies to the test antigen, they bindto the immobilized antigen in the detection window where they will bevisualized as two pink lines by binding of the detection reagent. Asingle pink line indicates that there is no antibody in the sample andthat the detection reagent is bound to immobilized, controlimmunoglobulin (Ig) in the detection window. The absence of any pinklines is indicative of a defective test.

The DAT test was performed essentially as described by Sundar and Rai inClin Diag Lab Immunol 9: 951-958, 2002.

The results of this analysis demonstrated that K28 identified asignificantly greater number of cases of human VL than did K39 alone.More specifically, K28 provided increased sensitivity in humans (66/69samples, 95.6%) to detect visceral leishmaniasis versus K39 alone (61/69samples, 88.4%) when employed in a dual path rapid test format. Inaddition, 4 patient samples with very low DAT titers (<3200) werecorrectly identified by the K28 rapid test, while only 2 of the 4 werecorrectly identified by the K39 rapid test.

These and other changes can be made to the embodiments in light of theabove-detailed description. In general, in the following claims, theterms used should not be construed to limit the claims to the specificembodiments disclosed in the specification and the claims, but should beconstrued to include all possible embodiments along with the full scopeof equivalents to which such claims are entitled. Accordingly, theclaims are not limited by the disclosure.

The various embodiments described above can be combined to providefurther embodiments. All of the U.S. patents, U.S. patent applicationpublications, U.S. patent applications, foreign patents, foreign patentapplications and non-patent publications referred to in thisspecification and/or listed in the Application Data Sheetareincorporated herein by reference, in their entirety. Aspects of theembodiments can be modified, if necessary to employ concepts of thevarious patents, applications and publications to provide yet furtherembodiments.

1. An isolated fusion polypeptide comprising: (i) the Leishmania K26sequence set forth in SEQ ID NO: 5; (ii) the Leishmania K39 sequence setforth in SEQ ID NO: 6; and (iii) the Leishmania K9 sequence set forth inSEQ ID NO:
 7. 2. The fusion polypeptide of claim 1, wherein the fusionpolypeptide comprises the amino acid sequence as set forth in amino acidresidues 10-262 of SEQ ID NO:
 8. 3. The fusion polypeptide of claim 1,wherein the fusion polypeptide further comprises an N-terminal aminoacid sequence of MHHHHHHTS (SEQ ID NO: 21).
 4. A diagnostic kit fordetecting asymptomatic or sub-clinical visceral leishmaniasis in abiological sample selected from the group consisting of sera, blood, andsaliva, comprising: (a) a fusion polypeptide of claim 1; and (b) adetection reagent.
 5. A diagnostic kit for identifying a patientafflicted with asymptomatic or sub-clinical visceral leishmaniasis thatis likely to develop acute visceral leishmaniasis, comprising: (a) afirst polypeptide comprising a fusion polypeptide of claim 1; (b) asecond polypeptide comprising an amino acid sequence as set forth in SEQID NOs: 10 or 11; and (c) a detection reagent.
 6. The kit of any ofclaim 4 or 5, wherein the detection reagent comprises a reporter groupconjugated to a binding agent.
 7. The kit of claim 6, wherein thebinding agent is selected from the group consisting ofanti-immunoglobulin, Protein G, Protein A and lectins.
 8. The kit ofclaim 6, wherein the reporter group is selected from the groupconsisting of radioisotopes, fluorescent groups, luminescent groups,enzymes, biotin and dye particles.
 9. An isolated fusion polypeptidewhich comprises the amino acid sequence set forth in 10-262 of SEQ IDNO: 8, or a sequence having at least 95% identity thereto along itsentire length.
 10. An isolated fusion polypeptide which comprises theamino acid sequence set forth SEQ ID NO: 8, or a sequence having atleast 95% identity thereto along its entire length.